In vivo and in vitro matured bovine oocytes present a distinct pattern of single-cell gene expression.

Oocyte gene expression is a well controlled event that promotes gamete competence to undergo maturation, fertilization, and to support early embryo development, directly affecting reproductive outcomes. Considering that in vivo controlled ovarian stimulation or in vitro maturation (IVM) for the acquisition of mature oocytes has distinct implications for gene expression, we sought to evaluate the effects of these procedures on the expression of competence-related genes in single-cell oocytes. Healthy Nelore cows of reproductive age were synchronized to harvest in vivo matured oocytes; ovaries from slaughtered animals were used to obtain cumulus-oocyte complexes that were in vitro matured. Single-cell gene expression was performed using TaqMan Low-Density Arrays and 42 genes were evaluated. In silico analysis of protein interactions and Gene Ontology (GO) analysis was performed. Reduced gene expression was observed for 24 targets in IVM oocytes when compared with those of in vivo matured oocytes (P < 0.05). Differences ranged from 1.5-fold to 4.8-fold higher in in vivo oocytes and the BMP15 (5.28), GDF9 (6.23), NOBOX (7.25), HSPA8 (7.85) and MSX1 (11.00) showed the greatest fold increases. The strongest score of functional interactions was observed between the CDC20 and CKS2, with the differentially expressed gene CDC20 being the main marker behind GO enrichment. IVM negatively affected the expression of important genes related to oocyte competency, and showed higher expression levels in in vivo matured oocytes. In vivo controlled ovarian stimulation may be a better strategy to achieve proper oocyte competence and increase the success of assisted reproductive technologies.

Reversible Contraception in Males: An Obtainable Target?

The last few decades have brought contraception to the forefront of research, with great strides made in effectively targeting and optimizing the physiology, pharmacology, and delivery processes that prevent pregnancy. However, these advances still predominantly target female contraceptives for the prevention of contraception, whereas targeting the male sex has lagged far behind. This has led to a marked deficiency in safe and effective male contraceptive agents, resulting in a heavy dependence on female contraceptives to prevent unwanted and unplanned pregnancies. Current research in the veterinary field and in rodents highlights several promising avenues whereby novel, safe, and effective male contraceptive alternatives are being developed-with an emphasis on reduced side effects and reversibility potential. This review aims to discuss current and novel male contraceptives (both human and veterinary formulations) while highlighting their efficacy, advantages, and disadvantages.

Supplementation with small-extracellular vesicles from ovarian follicular fluid during in vitro production modulates bovine embryo development.

Pregnancy success results from the interaction of multiple factors, among them are folliculogenesis and early embryonic development. Failure during these different processes can lead to difficulties in conception. Alternatives to overcome these problems are based on assisted reproductive techniques. Extracellular vesicles are cell-secreted vesicles present in different body fluids and contain bioactive materials, such as messenger RNA, microRNAs (miRNAs), and proteins. Thus, our hypothesis is that extracellular vesicles from follicular fluid from 3-6 mm ovarian follicles can modulate bovine embryo development in vitro. To test our hypothesis follicular fluid from bovine ovaries was aspirated and small-extracellular vesicles (<200 nm) were isolated for further analysis. Additionally, small-extracellular vesicles (EVs) were utilized for functional experiments investigating their role in modulating messenger RNA, microRNA as well as global DNA methylation and hydroxymethylation levels of bovine blastocysts. EVs from 3-6 mm follicles were used for RNA-seq and miRNA analysis. Functional annotation analysis of the EVs transcripts revealed messages related to chromatin remodeling and transcriptional regulation. EVs treatment during oocyte maturation and embryo development causes changes in blastocyst rates, as well as changes in the transcription levels of genes related to embryonic metabolism and development. Supplementation with EVs from 3-6 mm follicles during oocyte maturation and early embryo development (until the 4-cell stage) increased the levels of bta-miR-631 (enriched in EVs from 3-6 mm follicles) in embryos. Interestingly, the addition of EVs from 3-6 mm follicles induced changes in global DNA methylation and hydroxymethylation levels compared to embryos produced by the standard in vitro production system. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of 3-6 mm follicles during oocyte maturation and early embryo development can partially modify metabolic and developmental related genes as well as miRNA and global DNA methylation and hydroxymethylation, suggesting that EVs play an important role during oocyte maturation and early embryo development in vitro.

Plasma steroid dynamics in late- and near-term naturally and artificially conceived bovine pregnancies as elucidated by multihormone high-resolution LC-MS/MS.

The plasma levels of corticosteroids and sex steroids during pregnancy are key indicators of mammalian placental function and the onset of parturition. Steroid hormones are believed to be disturbed in pregnancies produced using assisted reproductive technologies (ARTs) due to placental dysfunction and the frequently observed lack of parturition signals. To elucidate the plasma steroid dynamics, a liquid chromatography-tandem mass spectrometry method was developed and used to determine the levels of corticosteroids (corticosterone, 11-deoxycortisol, and cortisol) and their direct precursors (progesterone and 17α-OH-progesterone) as well as sex steroids (androstenedione, estrone, estrone sulfate, testosterone, and 17ß-estradiol) in bovine plasma. The levels of these 10 steroids in recipient cows carrying naturally conceived (control), in vitro fertilized (IVF), or cloned (somatic cell nuclear transfer) conceptuses were compared during late-term pregnancy (30 days before parturition), during near-term pregnancy (1 day before parturition), and on the day of parturition (day 0). Significant differences were observed among the corticosteroid levels: higher levels of corticosterone, 11-deoxycortisol, and cortisol were detected in cloned pregnancies at day 30; lower levels of corticosterone were observed in ART-derived pregnancies at days 1 and 0; and estrone and estradiol levels were higher in IVF pregnancies throughout the final development. These results suggested an upregulation of the P450C11 and P450C21 enzymes 30 days before parturition in somatic cell nuclear transfer pregnancies and an overactivation of the aromatase enzyme in IVF pregnancies. Taken together, the monitoring of multiple steroid hormones revealed that the pregnancies obtained using ART exhibited plasma steroid concentration dynamics compatible with the dysregulation of steroidogenic tissues.

Follicle and corpus luteum size and vascularity as predictors of fertility at the time of artificial insemination and embryo transfer in beef cattle / Tamanho e vascularização do folículo e corpo lúteo como preditores de fertilidade após inseminação artificial e transferência de embriões em gado de corte

Two ultrasound based fertility prediction methods were tested prior to embryo transfer (ET) and artificial insemination (AI) in cattle. Female bovines were submitted to estrous synchronization prior to ET and AI. Animals were scanned immediately before ET and AI procedure to target follicle and corpus luteum (CL) size and vascularity. In addition, inseminated animals were also scanned eleven days after insemination to target CL size and vascularity. All data was compared with fertility by using gestational diagnosis 35 days after ovulation. Prior to ET, CL vascularity showed a positive correlation with fertility, and no pregnancy occurred in animals with less than 40% of CL vascularity. Prior to AI and also eleven days after AI, no relationship with fertility was seen in all parameters analyzed (follicle and CL size and vascularity), and contrary, cows with CL vascularity greater than 70% exhibit lower fertility. In inseminated animals, follicle size and vascularity was positive related with CL size and vascularity, as shown by the presence of greater CL size and vascularity originated from follicle with also greater size and vascularity. This is the first time that ultrasound based fertility prediction methods were tested prior to ET and AI and showed an application in ET, but not in AI programs. Further studies are needed including hormone profile evaluation to improve conclusion.

Duas técnicas de predição de fertilidade, baseadas em ultrassonografia, foram testadas no momento da transferência de embriões (TE) e inseminação artificial (IA) em bovinos. Fêmeas bovinas foram submetidas a protocolos de sincronização de estro para TE e IA. Os animais foram escaneados por ultrassonografia imediatamente antes do procedimento de TE e IA para identificar o tamanho e vascularização do folículo e corpo lúteo (CL). Além disso, os animais inseminados foram escaneados onze dias após a inseminação para identificar o tamanho e vascularização do CL. Todos os dados foram comparados com a fertilidade utilizando-se do diagnóstico gestacional 35 dias após a ovulação. No momento da TE, a vascularização do CL apresentou-se positivamente relacionada com a fertilidade, sendo que animais com menos de 40% de vascularização do CL não ficaram gestantes. No momento da IA, assim como onze dias após a IA, nenhuma relação foi encontrada entre fertilidade e os parâmetros analizados (tamanho e vascularização do folículo e CL), enquanto que contrariamente, houve uma queda na fertilidade em vacas com vascularização do CL acima de 70%. Nos animais inseminados, o tamanho e vascularização do folículo foi positivamente relacionado ao tamanho e vascularização do CL, demonstrado pela presença de um CL maior e mais vascularizado proveniente de um folículo maior e mais vascularizado. O presente estudo é o primeiro a mostrar métodos de predição de fertilidade baseados em ultrassonografia no momento da IA e TE, demonstrando uma aplicabilidade prática no momento da TE. Novos estudos são necessários para suportar os resultados mostrados, incluindo análises hormonais.

Correlations between testicular hemodynamic and sperm characteristics in rams / Correlações entre a hemodinâmica testicular e as características espermáticas em carneiros

The testicular artery is responsible for the blood supply that reaches the testis and has great importance in heat radiation. Vascular changes in the testis may lead to damage in sperm production, reflected in sperm motility and morphology. The aim of the present study was to evaluate correlations between testicular vascularity and sperm characteristic. Eight adult Santa Ines rams showing different reproductive status were used. The testicular vascularity and sperm characteristics were evaluated fortnightly during 90 days. Color Doppler ultrasonography was used to evaluate the testicular hemodynamic. Resistance index (RI) and pulsatility index (PI) of the testicular artery were evaluated by spectral-Doppler mode. The color-Doppler mode was used to evaluate the blood flow of the pampiniform plexus and testicular parenchyma. The semen analyses assessed were volume, concentration, motility, and morphology. The data were submitted to Pearson´s linear correlations test (p 0.05 was considered significant). No correlations were found between motility and testicular hemodynamic. The percentage of total sperm defects was positively correlated to left and right parenchymal score and to left RI and PI. On the other hand, the pampiniform plexus score was positively correlated to the number of colored pixels and negatively correlated to the RI and PI, for both sides. This study showed that the increase of sperm defect can be related to increase of testicular blood flow; however, more studies are need.

A artéria testicular é responsável pelo fluxo de sangue que chega aos testículos e tem grande importância na termorregulação. Mudanças vasculares nos testículos podem levar à queda da produção espermática, refletindo na motilidade e morfologia. O objetivo deste trabalho foi avaliar as correlações entre a vascularização testicular e as características espermáticas. Foram utilizados oito carneiros adultos Santa Inês com diferentes status reprodutivos. A vascularização testicular e as características seminais foram avaliadas por um período de 90 dias. A ultrassonografia Doppler colorida foi utilizada para avaliar a hemodinâmica testicular. Foram calculados os índices de resistência (RI) e os índices de pulsatilidade (PI) com o modo espectral do Doppler. O modo colorido do Doppler foi utilizado para analisar o fluxo sanguíneo do plexo pampiniforme e do parênquima testicular. As características seminais avaliadas foram o volume, concentração, motilidade e morfologia. Os dados foram submetidos ao teste de correlação linear de Person (P 0.05 foi considerado significativo). Não foram encontradas relações entre motilidade e a hemodinâmica testicular. A porcentagem de defeitos totais correlacionou-se positivamente com o escore de vascularização dos parênquimas direito e esquerdo, e com o RI e PI esquerdos. Também o escore de vascularização dos plexos se correlacionou positivamente com a média de pixels e negativamente com o RI e PI, de ambos os lados. Este trabalho mostrou que o aumento de defeitos espermáticos pode estar correlacionado com o aumento do fluxo sanguíneo nos testículos; contudo, mais estudos são necessários.