Lab partners: oocytes, embryos and company. A personal view on aspects of oocyte maturation and the development of monozygotic twins

The present review addresses the oocyte and the preimplantation embryo, and is intended to highlight the underlying principle of the "nature versus/and nurture" question. Given the diversity in mammalian oocyte maturation, this review will not be comprehensive but instead will focus on the porcine oocyte. Historically, oogenesis was seen as the development of a passive cell nursed and determined by its somatic compartment. Currently, the advanced analysis of the cross-talk between the maternal environment and the oocyte shows a more balanced relationship: Granulosa cells nurse the oocyte, whereas the latter secretes diffusible factors that regulate proliferation and differentiation of the granulosa cells. Signal molecules of the granulosa cells either prevent the precocious initiation of meiotic maturation or enable oocyte maturation following hormonal stimulation. A similar question emerges in research on monozygotic twins or multiples: In Greek and medieval times, twins were not seen as the result of the common course of nature but were classified as faults. This seems still valid today for the rare and until now mainly unknown genesis of facultative monozygotic twins in mammals. Monozygotic twins are unique subjects for studies of the conceptus-maternal dialogue, the intra-pair similarity and dissimilarity, and the elucidation of the interplay between nature and nurture. In the course of in vivo collections of preimplantation sheep embryos and experiments on embryo splitting and other microsurgical interventions we recorded observations on double blastocysts within a single zona pellucida, double inner cell masses in zona-enclosed blastocysts and double germinal discs in elongating embryos. On the basis of these observations we add some pieces to the puzzle of the post-zygotic genesis of monozygotic twins and on maternal influences on the developing conceptus.(AU)

Analysis of nuclear maturation, DNA damage and repair gene expression of bovine oocyte and cumulus cells submitted to ionizing radiation'

Radiotherapy causes destruction of tumor cells, but also threatens the integrity and survival of surrounding normal cells. Then, woman submitted to irradiation for cancer treatment may present permanent ovary damage, resulting in impaired fertility. The objective of this study was to investigate the effects of therapeutic doses of ionizing radiation (IR), used for ovarian cancer treatment in humans, on bovine cumulus-oocyte complexes (COCs) as experimental model. Bovine ovaries were exposed to 0.9 Gy, 1.8 Gy, 3.6 Gy or 18.6 Gy IR, and then COCs were collected and used to evaluate: (a) oocyte nuclear maturation; (b) presence of phosphorylated H2A.X (γH2AX), as an indicator of DNA double-strand breaks (DSBs); and (c) expression of genes involved in DNA repair (TP53BP1, RAD52, ATM, XRCC6 and XRCC5) and apoptosis (BAX). The radiation doses tested in this study had no detrimental effects on nuclear maturation and did not increase γH2AX in the oocytes. However, IR treatment altered the mRNA abundance of RAD52 (RAD52 homolog, DNA repair protein) and BAX (BCL2-associated X protein). We conclude that although IR doses had no apparent effect on oocyte nuclear maturation and DNA damage, molecular pathways involved in DNA repair and apoptosis were affected by IR exposure in cumulus cells.(AU)

Gonadal characterization of the Amazonian fish Serrapinnus kriegi (Characidae: Cheirodontinae) / Caracterização gonadal do peixe Amazônico Serrapinnus kriegi (Characidae: Cheirodontinae)

Abstract The knowledge of the testicular and ovarian morphology of a particular fish species is of paramount importance. Such analyze enables the development of studies and techniques aiming the improvement of their reproduction, management, commercialization and even their conservation. This study performed the ovarian and testicular characterization of the ornamental Amazon fish Serrapinnus kriegi. A total of three males and three females had their gonads analyzed by optical microscopy. Females present ovaries filled with oocytes in asynchronous development, indicating partial spawning in the species. Moreover, the micropyle and micropilar cell formation was observed in primary growing oocytes, representing a precocious oocyte development; and the zona radiata in the final vitellogenic oocytes is thicker than other related species, evidencing the development of a better protection to the embryos in function of the waters' turbulence that characterize it spawning sites in the Amazonian streams. The male specimens' present anastomosed tubular testes with unrestricted spermatogonia spread along the entire seminiferous tubules. The present data elucidate the dynamic of spermatogenesis and oogenesis of an ornamental Amazonian species, through the description of the male and female germ cells development.

Resumo O conhecimento da morfologia testicular e ovariana de uma determinada espécie de peixe é de suma importância, pois através destas análises é possível o desenvolvimento de estudos e técnicas visando o melhoramento de sua reprodução, manejo e comercialização e até mesmo auxiliar em sua conservação. Este estudo realizou a caracterização ovariana e testicular do peixe Amazônico ornamental Serrapinnus kriegi. Um total de três machos e três fêmeas tiveram suas gônadas analisadas através de microscopia óptica. As fêmeas apresentam ovários preenchidos por oócitos em desenvolvimento assincrônico, indicando desova parcelada da espécie. Além disso, observou-se a formação de micrópila e célula micropilar em oócitos em crescimento primário, representando o desenvolvimento precoce do oócito; a zona radiata nos oócitos vitelogênicos finais é mais espessa em comparação a outras espécies relacionadas, evidenciando o desenvolvimento de uma melhor proteção aos embriões, em função das águas turbulentas que caracterizam seu local de desova nos córregos amazônicos. Os machos apresentam testículos do tipo tubular anastomosado com espermatogônias irrestritas, espalhadas por todo o túbulo seminífero. Os dados apresentados elucidam a dinâmica da espermatogênese e oogênese de uma espécie de peixe ornamental amazônica, por meio da descrição das células germinativas masculinas e femininas.

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)

Lipopolysaccharide effects on activation and apoptosis of primordial ovarian follicles in heifers / Efeitos de lipopolissacarídeos na ativação e apoptose de folículos ovarianos primordiais em novilhas

The objective of the present study was to evaluate the effect of lipopolysaccharide (LPS) administration on activation and apoptosis of primordial follicles. There was no difference in the total number of follicles as well as in the different types of follicles. Furthermore, the LPS challenge didn't modulate the expression of genes related with ovarian reserve (HAM), oocyte survival (Survivin), activation rate (Pten, KIT, KITL1, KITL2, AKT1, SIRT1), and follicular abnormalities. Therefore, the LPS exposure with 24h interval had no effect on activation rate and primordial follicles abnormalities, and also had no effect on expression of anti-apoptotic genes and genes related with ovarian reserve, oocyte survival, activation rate, and primordial follicles abnormalities.

O objetivo do presente estudo foi avaliar o efeito da administração de lipopolissacarídeo (LPS) na ativação e a apoptose de folículos primordiais. Dez novilhas saudáveis (Bos taurus taurus), com idade média de 14 meses, alojadas em sistema de confinamento e alimentadas com TMR, foram utilizadas neste experimento. Os animais foram distribuídos aleatoriamente em dois grupos: grupo LPS (LPS; n = 5), que recebeu duas injeções intravenosas de 0,5µg/kg de peso corporal de lipopolissacarídeo (Sigma Aldrich®) diluído em 2mL de solução salina (0,9% de NaCl), com intervalo de 24h; e grupo controle (CTR; n = 5), que recebeu duas injeções intravenosas de 2mL de solução salina (0,9% de NaCl), com intervalo de 24h. A primeira injeção de LPS foi realizada no d 1, e no d 5 os animais foram abatidos, os ovários foram pesados e as amostras dos ovários foram coletadas para avaliação histológica e molecular. Não houve diferença no número total de folículos, bem como nos diferentes tipos de folículos. Além disso, o desafio com LPS não modulou a expressão de genes relacionados à reserva ovariana (HAM), à sobrevivência oocitária (Survivin), à taxa de ativação (Pten, KIT, KITL1, KITL2, AKT1, SIRT1) e às anormalidades foliculares. Portanto, a exposição ao LPS com intervalo de 24h não teve efeito sobre a taxa de ativação e as anormalidades dos folículos primordiais, bem como não teve efeito sobre a expressão de genes antiapoptóticos e de genes relacionados com a reserva ovariana, a sobrevivência oocitária, a taxa de ativação e as anormalidades dos folículos primordiais.

Insulin-like growth factor-1 improves in vitro meiotic resumption of dromedary camel (Camelus Dromedarius) oocytes

Despite relatively high maturation rate of in vitro matured oocytes in the dromedary camel, however, blastocyst production is very low after in vitro fertilization (IVF). Herein, the influences of oocyte collection method (follicular aspiration vs slicing; Experiment I), the addition of Insulin-like growth factor I (IGF-I) to the maturation medium (Experiment II) on in vitro maturation (IVM) of oocyte were investigated. Although the nuclear maturation did not differ regardless of collecting method, follicular aspiration led to lower degeneration rates than those in controls (P < 0.05). The percentages of oocytes at MII were greater in the presence of IGF-1 than in its absence (71.9% vs 48.4%, respectively, P<0.05). Additionally, the percentages of degenerated oocytes were higher in the control group compared to oocytes cultured in the presence of IGF-I (23.6% vs 10.4%, respectively, P<0.05). IGF-I treatment improved the quality of MII matured oocytes as evidenced by the decrease of cathepsin B (CTSB) activity, a marker of poor quality oocytes, when compared to control ones (P < 0.05). In conclusion, follicular aspiration decreased the degeneration rate; however, it had no effect on completion of maturation. IGF-I enhanced the IVM of oocyte and decreased degeneration rate.(AU)

Gonadal characterization of the Amazonian fish Serrapinnus kriegi (Characidae: Cheirodontinae)

Abstract The knowledge of the testicular and ovarian morphology of a particular fish species is of paramount importance. Such analyze enables the development of studies and techniques aiming the improvement of their reproduction, management, commercialization and even their conservation. This study performed the ovarian and testicular characterization of the ornamental Amazon fish Serrapinnus kriegi. A total of three males and three females had their gonads analyzed by optical microscopy. Females present ovaries filled with oocytes in asynchronous development, indicating partial spawning in the species. Moreover, the micropyle and micropilar cell formation was observed in primary growing oocytes, representing a precocious oocyte development; and the zona radiata in the final vitellogenic oocytes is thicker than other related species, evidencing the development of a better protection to the embryos in function of the waters turbulence that characterize it spawning sites in the Amazonian streams. The male specimens present anastomosed tubular testes with unrestricted spermatogonia spread along the entire seminiferous tubules. The present data elucidate the dynamic of spermatogenesis and oogenesis of an ornamental Amazonian species, through the description of the male and female germ cells development.

Resumo O conhecimento da morfologia testicular e ovariana de uma determinada espécie de peixe é de suma importância, pois através destas análises é possível o desenvolvimento de estudos e técnicas visando o melhoramento de sua reprodução, manejo e comercialização e até mesmo auxiliar em sua conservação. Este estudo realizou a caracterização ovariana e testicular do peixe Amazônico ornamental Serrapinnus kriegi. Um total de três machos e três fêmeas tiveram suas gônadas analisadas através de microscopia óptica. As fêmeas apresentam ovários preenchidos por oócitos em desenvolvimento assincrônico, indicando desova parcelada da espécie. Além disso, observou-se a formação de micrópila e célula micropilar em oócitos em crescimento primário, representando o desenvolvimento precoce do oócito; a zona radiata nos oócitos vitelogênicos finais é mais espessa em comparação a outras espécies relacionadas, evidenciando o desenvolvimento de uma melhor proteção aos embriões, em função das águas turbulentas que caracterizam seu local de desova nos córregos amazônicos. Os machos apresentam testículos do tipo tubular anastomosado com espermatogônias irrestritas, espalhadas por todo o túbulo seminífero. Os dados apresentados elucidam a dinâmica da espermatogênese e oogênese de uma espécie de peixe ornamental amazônica, por meio da descrição das células germinativas masculinas e femininas.

Differentially methylated regions identified in bovine embryos are not observed in adulthood

The establishment of epigenetic marks during the reprogramming window is susceptible to environmental influences, and stimuli during this critical stage can cause altered DNA methylation in offspring. In a previous study, we found that low levels of sulphur and cobalt (low S/Co) in the diet offered to oocyte donors altered the DNA methylome of bovine embryos. However, due to the extensive epigenetic reprogramming that occurs during embryogenesis, we hypothesized that the different methylation regions (DMRs) identified in the blastocysts may not maintain in adulthood. Here, we aimed to characterize DMRs previously identified in embryos, in the blood and sperm of adult progenies of two groups of heifers (low S/Co and control). We used six bulls and characterized the DNA methylation levels of KDM2A, KDM5A, KMT2D, and DOT1L genes. Our results showed that all DMRs analysed in both groups and tissues were hypermethylated unlike that noticed in the embryonic methylome profiles. These results suggest that embryo DMRs were reprogrammed during the final stages of de novo methylation during embryogenesis or later in development. Therefore, due to the highly dynamic epigenetic state during early embryonic development, we suggest that is essential to validate the DMRs found in embryos in adult individuals.(AU)

Gonadal characterization of the Amazonian fish Serrapinnus kriegi (Characidae: Cheirodontinae) / Caracterização gonadal do peixe Amazônico Serrapinnus kriegi (Characidae: Cheirodontinae)

The knowledge of the testicular and ovarian morphology of a particular fish species is of paramount importance. Such analyze enables the development of studies and techniques aiming the improvement of their reproduction, management, commercialization and even their conservation. This study performed the ovarian and testicular characterization of the ornamental Amazon fish Serrapinnus kriegi. A total of three males and three females had their gonads analyzed by optical microscopy. Females present ovaries filled with oocytes in asynchronous development, indicating partial spawning in the species. Moreover, the micropyle and micropilar cell formation was observed in primary growing oocytes, representing a precocious oocyte development; and the zona radiata in the final vitellogenic oocytes is thicker than other related species, evidencing the development of a better protection to the embryos in function of the waters’ turbulence that characterize it spawning sites in the Amazonian streams. The male specimens’ present anastomosed tubular testes with unrestricted spermatogonia spread along the entire seminiferous tubules. The present data elucidate the dynamic of spermatogenesis and oogenesis of an ornamental Amazonian species, through the description of the male and female germ cells development.

O conhecimento da morfologia testicular e ovariana de uma determinada espécie de peixe é de suma importância, pois através destas análises é possível o desenvolvimento de estudos e técnicas visando o melhoramento de sua reprodução, manejo e comercialização e até mesmo auxiliar em sua conservação. Este estudo realizou a caracterização ovariana e testicular do peixe Amazônico ornamental Serrapinnus kriegi. Um total de três machos e três fêmeas tiveram suas gônadas analisadas através de microscopia óptica. As fêmeas apresentam ovários preenchidos por oócitos em desenvolvimento assincrônico, indicando desova parcelada da espécie. Além disso, observou-se a formação de micrópila e célula micropilar em oócitos em crescimento primário, representando o desenvolvimento precoce do oócito; a zona radiata nos oócitos vitelogênicos finais é mais espessa em comparação a outras espécies relacionadas, evidenciando o desenvolvimento de uma melhor proteção aos embriões, em função das águas turbulentas que caracterizam seu local de desova nos córregos amazônicos. Os machos apresentam testículos do tipo tubular anastomosado com espermatogônias irrestritas, espalhadas por todo o túbulo seminífero. Os dados apresentados elucidam a dinâmica da espermatogênese e oogênese de uma espécie de peixe ornamental amazônica, por meio da descrição das células germinativas masculinas e femininas.

Gonadal characterization of the Amazonian fish Serrapinnus kriegi (Characidae: Cheirodontinae) / Caracterização gonadal do peixe Amazônico Serrapinnus kriegi (Characidae: Cheirodontinae)

The knowledge of the testicular and ovarian morphology of a particular fish species is of paramount importance. Such analyze enables the development of studies and techniques aiming the improvement of their reproduction, management, commercialization and even their conservation. This study performed the ovarian and testicular characterization of the ornamental Amazon fish Serrapinnus kriegi. A total of three males and three females had their gonads analyzed by optical microscopy. Females present ovaries filled with oocytes in asynchronous development, indicating partial spawning in the species. Moreover, the micropyle and micropilar cell formation was observed in primary growing oocytes, representing a precocious oocyte development; and the zona radiata in the final vitellogenic oocytes is thicker than other related species, evidencing the development of a better protection to the embryos in function of the waters turbulence that characterize it spawning sites in the Amazonian streams. The male specimens present anastomosed tubular testes with unrestricted spermatogonia spread along the entire seminiferous tubules. The present data elucidate the dynamic of spermatogenesis and oogenesis of an ornamental Amazonian species, through the description of the male and female germ cells development.(AU)

O conhecimento da morfologia testicular e ovariana de uma determinada espécie de peixe é de suma importância, pois através destas análises é possível o desenvolvimento de estudos e técnicas visando o melhoramento de sua reprodução, manejo e comercialização e até mesmo auxiliar em sua conservação. Este estudo realizou a caracterização ovariana e testicular do peixe Amazônico ornamental Serrapinnus kriegi. Um total de três machos e três fêmeas tiveram suas gônadas analisadas através de microscopia óptica. As fêmeas apresentam ovários preenchidos por oócitos em desenvolvimento assincrônico, indicando desova parcelada da espécie. Além disso, observou-se a formação de micrópila e célula micropilar em oócitos em crescimento primário, representando o desenvolvimento precoce do oócito; a zona radiata nos oócitos vitelogênicos finais é mais espessa em comparação a outras espécies relacionadas, evidenciando o desenvolvimento de uma melhor proteção aos embriões, em função das águas turbulentas que caracterizam seu local de desova nos córregos amazônicos. Os machos apresentam testículos do tipo tubular anastomosado com espermatogônias irrestritas, espalhadas por todo o túbulo seminífero. Os dados apresentados elucidam a dinâmica da espermatogênese e oogênese de uma espécie de peixe ornamental amazônica, por meio da descrição das células germinativas masculinas e femininas.(AU)

Mecanismos epigenéticos nos espermatozoides e o impacto sobre a fertilidade masculina / Epigenetic mechanisms on sperm cells and the impact on male fertility

Por ser uma célula altamente especializada, o espermatozoide apresenta diferentes mecanismos epigenéticos, sendo os principais as metilações do DNA, o código de histonas, os ncRNAs (RNAs não codificadores), e a alta condensação da cromatina pela presença das protaminas. Estes mecanismos interagem entre si, contribuindo para a formação do epigenoma espermático, que modela a carga molecular espermática, que, por sua vez, pode impactar sobre as características do desenvolvimento embrionário e da progênie. Dessa forma, atualmente é consenso que o papel do espermatozoide ultrapassa a entrega de DNA de qualidade para o oócito no momento da fecundação. Pesquisas recentes de diversos grupos, incluindo o nosso, mostram que além da contribuição com DNA de qualidade, o espermatozoide entrega moléculas ao oócito no momento da fecundação que influenciam o desenvolvimento do embrião. Recentemente, essas moléculas de origem espermática (Em inglês: sperm-borne) também são associadas com alterações metabólicas e cognitivas da progênie. Embora ainda pouco se entenda como esses mecanismos podem persistir mesmo com o ciclo de reprogramação celular que ocorre logo após a fecundação, é evidente que estes podem impactar as características da progênie. Nesta revisão abordaremos sobre a modulação do epigenoma espermático e seus efeitos no desenvolvimento embrionário.(AU)

Since it is a highly specialized cell, the spermatozoa display different epigenetic mechanisms; the main ones are DNA methylation, histone code, ncRNAs (non-coding RNAs), and high chromatin condensation by the presence of protamines. These mechanisms act in synergy contributing to forming the sperm epigenome, which modulates the spermatic molecular cargo, and, may impact embryo and offspring development features. Thus, it is currently a consensus that the role of spermatozoa goes beyond delivering quality DNA to the oocyte at fertilization. Relevant findings from several research groups, including ours, have shown that sperm delivers several molecules to the oocyte at fertilization, beyond the contribution to DNA, which influences the development of the embryo. Recently, these sperm-borne molecules have also been associated with metabolic and cognitive changes in the offspring. Although the mechanism by which these changes can persist even after embryo reprogramming is not completely understood, evidence shows that sperm cell molecular content impacts embryo and offspring development. This review will mainly focus on the modulation of the sperm epigenome and its effects on embryo development.(AU)

The waveless model: clinical and research applications of follicular wave suppression in cattle / Modelo waveless: aplicações clínicas e na pesquisa da supressão das ondas foliculares em bovinos

The follicular growth waves are directly linked to the fluctuations in plasma gonadotrophins, which are controlled by the hypothalamic GnRH release pattern. Therefore, if the actions of the GnRH are inhibited or blocked, the final stages of the antral follicle growth are suppressed, resulting in an induced anestrus (a.k.a. waveless model). In the human medicine, GnRH agonists or antagonists are broadly used in the control of ovarian disfunctions, as well as in the preparation of women for assisted reproductive cycles. In cattle, a similar effect can be obtained by active immunization against GnRH. This was shown to be a viable strategy, for example, for the control of chronic cases of cystic ovarian disease in oocyte donors. However, on shall take into account the substantial individual variation on the immune response and, consequently, the lack of control of the duration of the anestrus induced. The waveless model is also very useful as a research model, once it controls the potential interference of the endogenous FSH and LH, improving the sensitivity of essays with exogenous hormones and consequently reducing the required number of replicas within studies.(AU)

O padrão de crescimento folicular em ondas está diretamente associado às flutuações nas concentrações plasmáticas de gonadotrofinas, controladas por sua vez pelo padrão de liberação de GnRH hipotalâmico. Desta forma, a inibição ou bloqueio da ação do GnRH suprime as etapas finais do crescimento folicular, resultando em anestro induzido (também chamado modelo waveless). Na medicina humana, agonistas ou antagonistas de GnRH são utilizados tanto no controle de disfunções ovarianas quanto na preparação de pacientes para procedimentos de reprodução assistida. Em bovinos, este efeito pode ser obtido pela imunização ativa contra GnRH, e mostrou-se estratégia viável, por exemplo, no controle de casos crônicos de doença ovariana cística em doadoras de oócitos. Contudo, é importante considerar a grande variação individual na resposta à imunização e consequente impossibilidade de controlar a duração do anestro induzido. O modelo waveless também é de grande utilidade na pesquisa, uma vez que elimina a potencial interferência do FSH e LH endógenos, aumentando a sensibilidade nos ensaios com hormônios exógenos e consequentemente reduzindo o número de réplicas necessárias nos estudos.(AU)

Pregnancy success in bitches - evaluation of interactions between artificial insemination method, serum progesterone concentration and vaginal cytology parameters

Background: The artificial insemination has become a well-established method in the breeding of bitches, and evaluation of the factors that may potentially affect pregnancy success is essential. For this reason, it is essential to evaluate the factors that may affect fertility of the bitch when artificial insemination is performed. Serum progesterone concentrations and vaginal cytology have been used to determine the time of ovulation and stage of the estrus cycle. This study aimed to evaluate the artificial insemination method, the serum progesterone concentration, the breed size, age, the whelping number, vaginal cytology parameters, and their interactions on pregnancy success in bitches. Materials, Methods & Results: A total of 607 bitches that had undergone reproductive consultation with the Mexican Canine Federation from January to December 2016 were enrolled in the present study and assigned to one of 2 artificial insemination methods (intravaginal and transcervical) using fresh semen. Determination of the estrus cycle phase and the time of Artificial insemination was based on vaginal cytology and serum progesterone concentrations. Bitches inseminated by the transcervical technique had a higher pregnancy rate with respect to females inseminated by the intravaginal technique (P < 0.05). Moreover, females with a serum progesterone concentration of 5-10 ng/mL had a greater probability (> 4 times) of getting pregnant than animals with lower or higher progesterone concentrations (P < 0.05). Bitches inseminated by the intravaginal technique and with serum progesterone concentrations >10 ng/mL had a considerable reduction in pregnancy (P < 0.05) compared with females with < 10 ng/mL serum progesterone or with bitches inseminated by the transcervical technique. Discusion: Serum progesterone concentration, the artificial insemination method, and superficial cells without a nucleus modified the pregnancy rate in bitches. Females inseminated by transcervical semen deposition had a higher pregnancy rate than females inseminated by the intravaginal technique. Using fresh or frozen-thawed semen produced a higher pregnancy rate in bitches inseminated by transcervical semen deposition than females inseminated by the intravaginal technique. Differences in the pregnancy rate between transcervical and intravaginal insemination could be associated with the correct semen disposition, the distance that the sperm must travel to reach the oocyte, as well as the number of sperm that reach the oviduct ampulla. Exist evidences that after ovulation, as progesterone rises, the cervix is closed, which may compromise the passage of the sperm deposited into the vagina. Therefore, it is likely that in females with a serum progesterone concentration > 10 ng/mL, the cervix was closed, compromising the ability of the sperm to access the oviduct. Thus, the use of intravaginal insemination should be done in bitches with a serum progesterone concentrations < 11 ng/mL to reduce the possibility of cervical closure and to increase the odds of pregnancy. It is well documented that the serum progesterone concentration and vaginal cytology parameters have a great influence on pregnancy success, and the results confirm these findings. In the present study, 96% of the bitches inseminated with a serum progesterone concentration of 5-10 ng/mL got pregnant and had higher odds of pregnancy than bitches with lower or higher serum progesterone concentrations.

Comparison of efficiency between two artificial insemination methods using fresh semen in domestic cat (Felis Catus) / Comparação da eficiência entre dois métodos de inseminação artificial utilizando sêmen fresco em gata doméstica (Felis Catus)

This study aimed to compare domestic cats' pregnancy rates using fresh semen for the bilateral intrauterine insemination (BIUI) method and the novel uterine body insemination (UBI) method. Queens received a single injection of equine chorionic gonadotropin (eCG) (200 IU; IM) to induce ovarian follicular development and, after 83 h, an injection of human chorionic gonadotropin (hCG) (100 IU; IM) for final oocyte maturation and ovulation induction. Thirty-four hours after hCG administration, 3 × 106fresh spermatozoa were used for insemination by the BIUI (n = 8 queens) or by the UBI (n = 7 queens) techniques,respectively. Pregnancy rates were 75.00% (6/8) by BIUI and 42.85% (3/7) by the UBI method. Themean litter size was 3.0 ± 0.86 for the BIUI, and 2.0 ± 1.0 for the UBI method. Spontaneous abortion occurred on day 35 of pregnancy in one queen following the UBI method. Our findings showed that the BIUI of queens with fresh semen resulted in higher pregnancy rates than the novel UBI method; also, acceptable pregnancy rates were achieved following BIUI with fresh semen in the domestic cat.(AU)

O objetivo deste estudo foi comparar as taxas de prenhez em gatas domésticas usando sêmen fresco para o método de inseminação intrauterina bilateral (BIUI) e o novo método de inseminação do corpo uterino (UBI). As gatas receberam uma única injeção de gonadotrofina coriônica equina (eCG) (200 UI; IM) para induzir o desenvolvimento folicular ovariano e, após 83 h, uma injeção de gonadotrofina coriônica humana (hCG) (100 UI; IM) para maturação final do oócito e ovulação indução. Trinta e quatro horas após a administração de hCG, 3 × 106 espermatozoides frescos foram utilizados para inseminação pelas técnicas de BIUI (n = 8 gatas) ou UBI (n = 7 gatas), respectivamente. As taxas de gravidez foram de 75,00% (6/8) pela BIUI e 42,85% (3/7) pelo método UBI. O tamanho médio da ninhada foi de 3,0 ± 0,86 para o método BIUI e 2,0 ± 1,0 para o método UBI. Aborto espontâneo ocorreu no dia 35 de gestação em uma gata seguindo o método UBI. Nossos achados mostraram que a BIUI de gatas com sêmen fresco resultou em maiores taxas de prenhez do que o novo método UBI; também, taxas de prenhez aceitáveis foram alcançadas após BIUI com sêmen fresco no gato doméstico.(AU)

The impact of microplastics on female reproduction and early life

Plastic pollution in our environment is one of the most important global health concerns right now. Micro- and nanoplastics (MNPs) are taken up by both humans and animals, mainly via food and water, and can pass important epithelial barriers. Indications of plastics in the blood circulation have recently been shown in both humans and farm animals, but standardized methods to quantify the exact levels of MNPs to which we are exposed are currently lacking. Potential hazards of MNPs are being investigated very recently, including the impact that MNPs may have on reproduction. However, studies on mammalian reproduction are scarce, but a wealth of data from aquatic species indicates reproductive effects of MNPs. The first studies in rodent models demonstrate that MNPs reach the gonads after oral exposure and may impact offspring after maternal exposure during the gestational period. These effects may arise from the particles themselves or the presence of plastic contaminants that leach from plastics. Plastic contamination has been detected in human placentas, fetal fluid and the meconium of newborns, indicating the presence of plastics from the very first start of life. Currently there is a lack of studies that investigate the impact of MNP exposure during the periconception and embryonic period, whereas this is an extremely sensitive period that needs considerable attention with the growing amount of plastics in our environment.(AU)

Interação da sazonalidade e nutrição na viabilidade embrionária ovina / Interaction between seasonality and nutrition on embrio viability of ovines

A sazonalidade reprodutiva e a condição nutricional são os principais fatores que influenciam o desempenho reprodutivo em ovinos, principalmente em áreas onde a disponibilidade de alimentos é altamente sazonal. A desnutrição pode comprometer a competência folículo-oocitária, a função lútea e o desenvolvimento embrionário. Por outro lado, o tratamento com melatonina é um método eficaz para induzir ciclicidade, aumentando a ovulação e melhorando a viabilidade do embrião durante o anestro. Portanto, levantamos a hipótese de que a melatonina pode substituir os efeitos prejudiciais da desnutrição para melhorar o desempenho reprodutivo em ovelhas. A este respeito, esta revisão enfoca os efeitos da interação entre melatonina e nutrição na viabilidade do embrião e resume a informação disponível sobre o efeito da melatonina exógena na viabilidade do embrião em ovelhas desnutridas.(AU)

Reproductive seasonality and nutritional condition are the main factors that influence reproductive performance in sheep, particularly in the areas where the availability of food is highly seasonal. Undernutrition can compromise follicle-oocyte competence, luteal function and embryo development. On the other hand, melatonin treatment is an effective method for inducing estrous cycles, increasing ovulation and improving embryo viability during anestrus. Therefore, we hypothesized that melatonin can override the detrimental effects of undernutrition to improve reproductive performance in ewes. In this regard, this review focuses on the effects of the interaction between melatonin and nutrition on embryo viability and summarizes the available information about the effect of exogenous melatonin on embryo viability in undernourished ewes.(au)

Criopreservação do sêmen suíno: desafios a serem superados que auxiliam a identificação de espermatozoides mais férteis / Boar semen cryopreservation: challenges to be overcome that help the identification of more fertile spermatozoa

A criopreservação é o melhor método de armazenamento de sêmen por período indeterminado, conferindo alta flexibilidade de uso. Além disso, é o principal meio para formação de banco de germoplasma. No entanto, congelar sêmen suíno ainda é um desafio a ser enfrentado. Isso porquê, além das características de membrana espermática que ocasionam maiores efeitos deletérios às células durante os processos de congelação e descongelação, ainda é preciso lidar com a grande variabilidade de resultados, seja entre indivíduos, ejaculados de um mesmo indivíduo ou ainda, frações de um mesmo ejaculado. Frente a isso, diferentes estratégias têm sido investigadas a fim de obter melhores resultados e assim, aumentar a aplicabilidade do sêmen congelado suíno a nível comercial. Além disso, acreditamos que haja a possibilidade de se relacionar a capacidade de criotolerância de um determinado espermatozoide a sua maior ou menor capacidade de fertilizar o oócito.(AU)

Cryopreservation is the best method of storing semen indefinitely, providing high flexibility of use. In addition, it is the primary means for forming a germplasm bank. However, freezing boar semen is still a challenge to be faced because, in addition to the characteristics of the spermatic membrane that cause deleterious effects to the cells during the freezing and thawing processes, it is still necessary to deal with the significant variability of results, whether between individuals, ejaculates from the same individual or even fractions of an even ejaculated. Because of this, different strategies have been investigated to obtain better results and thus increase the applicability of frozen boar semen at a commercial level. Furthermore, we believe it is possible to relate the cryotolerance capacity of a given sperm to its greater or lesser capacity to fertilize the oocyte.(AU)

Dynamics of nuclear and cytoplasm maturation of bovine oocytes cultivated in vitro in medium supplemented with fulerol / Dinâmica da maturação nuclear e citoplasmática de oócitos bovinos cultivados in vitro em meio suplementado com fulerol

The aim of this work was to evaluate, in vitro, the dynamics of nuclear and cytoplasmic maturation of bovine oocytes in traditional IVM medium (CT) and supplemented with fullerol (MF50), for 36 hours. The nuclear maturation of CT (n=300) and MF50 (n=270) every 6 hours, stained with Hoechst33342 and cytoplasmic, the mitochondrial distribution of CT (n=197) and MF50 (n=159) at every 12 hours, stained with Mitotracker Orange. At 6 hours, CT oocytes (19%) were in MI (metaphase I), while in MF50 they were in GV (germ vesicle) or GVB (GV breakeage), repeating at 12 hours. At 18 hours, 46.3% were matured in CT, and 20% in MF50. At 24 hours, 43.9% of maturation was observed in the MF50 group, and 63.8% in the CT. At 30 and 36 hours, the maturation pattern was stable, but with the onset of oocyte degeneration. There was a delay in cytoplasmic maturation with 36 hours (P < 0.05) in MF50 (53.9% of mature gametes), compared to CT (69.8%). With immature cytoplasm, they were 10.4% and 31.7% for CT and MF50 (P< 0.05), respectively. It was concluded that fullerol possibly interfered in the expansion of cumulus oophorus cells, as well as delayed the meiotic progression and cytoplasmic maturation.

O objetivo deste estudo foi avaliar, in vitro, a dinâmica da maturação nuclear e citoplasmática de oócitos bovinos em meio MIV tradicional (TC) e suplementado com fulerol (MF50), durante 36 horas. Na maturação nuclear do TC (n=300) e do MF50 (n=270) a cada seis horas, corados com Hoechst 33342, e na citoplasmática, avaliou-se a distribuição mitocondrial do TC (n=197) e do MF50 (n=159) a cada 12 horas, corados com Mitotracker Orange (Life® Technologies). Às seis horas, oócitos do TC (19%) se encontravam em MI (metáfase I), enquanto no MF50 estavam em VG (vesícula germinativa) ou QVG (quebra VG), repetindo com 12 horas. Às 18 horas, 46,3% estavam maturados no TC, e 20% no MF50. Com 24 horas, verificaram-se 43,9% de maturação no grupo MF50, e 63,8% no TC. Às 30 e 36 horas, o padrão de maturação foi estável, mas com início de degeneração oócitária. Houve retardo na maturação citoplasmática com 36 horas (P<0,05) no MF50 (53,9% de gametas maduros), comparado ao TC (69,8%). Com citoplasma imaturo, foram 10,4% e 31,7% para TC e MF50 (P<0,05), respectivamente. Conclui-se que o fulerol possivelmente interferiu na expansão das células do cumulus oophorus, bem como retardou a progressão meiótica e a maturação citoplasmática dos oócitos.

Effect of the presence and location of corpus luteum on competence of bovine cumulus-oocyte complexes

This study aimed to determine the effect of presence of the corpus luteum (CL) and its influence on cumulus­oocyte complexes (COCs) obtained from the ipsilateral or contralateral ovary in bovine on the recovery and capacity of the oocytes to sustain mono-spermic fertilization, undergo preimplantation development, and develop to the blastocyst stage. Ovaries were collected at a local slaughterhouse and kept in pairs corresponding to the same animal. In the first experiment the variables evaluated were compared between cows with (CCL+) and without (CCL- ) CL, and for the second experiment, comparisons were made between ovaries with an ipsilateral (CL+), contralateral (CL−), and no (NCL). The recovery rate of COCs was higher in ovaries from CCL− cows, and a higher proportion of grade 1 COCs were recovered from this group. A higher proportion of metaphase I oocytes at 7 h of maturation, and a higher rate of cleavage were observed in the CCL+ group; however, a higher proportion of embryos were obtained from the CCL− group. Besides, COCs from the CL+ group had a lower proportion of grades 1 and 2 morphological qualities, lower rate of metaphase II oocytes at 22 h of maturation, and lower rate of formation of two pronuclei, whereas a higher proportion of unfertilized oocytes after in vitro fertilization. On the other hand, the COCs from the CL− group displayed a lower proportion of oocytes with more than two pronuclei, higher cleavage rate, and higher final blastocyst production were obtained when compared to CL+. Thus, the effects of CL on the competence of bovine COCs are different depending on the anatomical proximity of their location in the animal, negatively affecting the quality of COCs located in the same ovary, but not having negative effects on the competence of COCs in the ovaries contralateral to their location.(AU)

Genetic evaluation of oocyte and embryo production in dairy Gir cattle using repeatability and random regression models

The objective of this work is to estimate genetic parameters and breeding values to improve embryo and oocyte production, using repeatability and random regression models (RRM) for Gir dairy cattle. We used 11,398 records of ovum pick-up from 1,747 dairy Gir donors and evaluated sixteen different models: the traditional repeatability model and fifteen RRM, each of which considered a different combination of Legendre polynomial regressors to describe the additive genetic and permanent environment effects. The 4G1P model (four regressors for the genetic effect and one regressor for the permanent environment effect) is the most suitable model to analyze the number of viable and total oocytes, while the 3G1P is the best model to analyze the number of cleaved and viable embryos, according to the values of the Akaike information criterion (AIC) and the Bayesian information criterion (BIC). The heritability estimated with the RRM was higher than that estimated with the repeatability model. The high repeatability reported for oocyte and embryo count traits indicates that donors, which had high oocyte and embryo counts in the first ovum pick-up, should maintain this result in the next ovum pick-up. Genetic correlations between adjacent ages were high and positive, while genetic correlations between extreme ages were weak. We observed a reranking of the top sires and females (heifers and cows) over the period evaluated. The reliability of the estimated breeding values by RRM showed changes across age, and the expected genetic gains by RRM are larger. This shows that RRM is most suitable alternative for the evaluation and selection of oocyte and embryo count traits.(AU)