Methylation and acetylation of histone H3 at lysine 27 (H3K27) regulate chromatin structure and gene expression during early embryo development. While H3K27 acetylation (H3K27ac) is associated with active gene expression, H3K27 methylation (H3K27me) is linked to transcriptional repression. The aim of this study was to assess the profile of H3K27 acetylation and methylation (mono-, di- and trimethyl) during oocyte maturation and early development in vitro of porcine embryos. Oocytes/embryos were fixed at different developmental stages from germinal vesicle to day 8 blastocysts and submitted to an immunocytochemistry protocol to identify the presence and quantify the immunofluorescence intensity of H3K27ac, H3K27me1, H3K27me2 and H3K27me3. A strong fluorescent signal for H3K27ac was observed in all developmental stages. H3K27me1 and H3K27me2 were detected in oocytes, but the fluorescent signal decreased through the cleavage stages and rose again at the blastocyst stage. H3K27me3 was detected in oocytes, in only one pronucleus in zygotes, cleaved-stage embryos and blastocysts. The nuclear fluorescence signal for H3K27me3 increased from the 2-cell stage to 4-cell stage embryos, decreased at the 8-cell and morula stages and increased again in blastocysts. Different patterns of the H3K27me3 mark were observed at the blastocyst stage. Our results suggest that changes in the H3K27 methylation status regulate early porcine embryo development as previously shown in other species.

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The goal of this study was to determine the distribution of pre-antral follicles in the ovarian parenchyma of mares. For Experiment 1, each ovary was cut longitudinally at the greater curvature, performing two hemiovaries. After that, six fragments from each hemiovary were obtained, resulting in 12 fragments, which were divided into the innermost region of the parenchyma, the middle region and the outermost region. All the three obtained sections were cut transversally to obtain two fragments from each one. For Experiment 2, each ovary also submitted to a longitudinal cut on the greater curvature, forming two hemiovaries. Each hemiovary was sectioned into four symmetrical fragments, resulting in eight fragments per ovary. The fragments were related as being near to or far from the ovulatory fossa. The fragments of both experiments were immediately fixed in Carnoy for 12 hr and kept in 70% ethanol for 24 hr. Follicles were classified according to the stages of development and for morphological integrity according to oocyte morphology and granulosa cells. After the histological assessment, a total of 1,130 follicles were visualized from Experiment 1, being 1,054 (93.3%) primordial follicles and 76 (4.7%) follicles in development. The innermost region had the highest percentage of pre-antral follicles compared to the other regions (p < .05). The middle and outermost regions showed higher percentages of intact primordial and developing follicles than the innermost region (p < .05). Considering Experiment 2, 938 follicles were found, being 894 (95.3%) primordial and 44 (4.7%) follicles in development. The region near the ovulatory fossa presented higher (58.7%; 551 of 938) follicular concentration compared to the region far from the ovulatory fossa (41.3%; 387 of 938; p < .05). As a conclusion, distribution of pre-antral follicles in the equine ovary has a specific pattern through the parenchyma. Also, the follicular integrity differed in the studied ovarian areas.

In follicular aspiration, physical aspects are of high significance for the technique to succeed, such as vacuum pressure, caliber of the needle and the way the follicular wall curettage is performed. The aim of this study was to investigate the recovery rate of equine oocytes aspirated by scraping of the follicular wall, testing different calibers of disposable needles, as well as the morphological evaluation of the cumulus oophorus complexes (COCs). Mares ovaries (n=447) obtained at a local slaughterhouse were transported to the laboratory in a thermal container (20 °C) and had the tunica albuginea and connective tissues dissected. The aspirated follicles had 10 to 25 mm in diameter, and 30x8 (21G 1 ¼) or 40x12 (18G 1 ½) needles were used for the aspiration, forming group A (G-A) and group B (G-B), respectively. In G-A and G-B, 480 and 548 follicles were aspirated, respectively. Under the stereomicroscope, the oocytes were evaluated according to the quality of the ooplasm and characteristics of the cumulus cells (grade I, II, III and denuded). The statistical analysis was performed using the Student's t-test, logistic regression and test of proportions, and differences were considered significant when P<0.05. There was no difference between recovery rates of groups G-A (66.5%; 330/496) and G-B (65.5%; 359/548 ResumoNa obtenção de oócitos para a espécie equina, aspectos físicos são de alta significância para o sucesso da técnica, como a pressão de vácuo e o calibre de agulha utilizado, além da forma como é realizada a raspagem da parede folicular. O objetivo deste estudo foi investigar o índice de recuperação de oócitos equinos aspirados pela técnica de raspagem da parede folicular, testando calibres distintos de agulhas descartáveis, assim como avaliação morfológica dos complexos cumulus oophorus (CCOs). Foram utilizados ovários de éguas (n=447), obtidos em abatedouro local, transportados ao laboratório em recipiente térmico (20 ºC) e submetidos à dissecação da túnica albugínea e tecidos conectores. Os folículos aspirados obtinham diâmetro entre 10 mm a 25 mm, e para tanto utilizou-se a agulha 30x8 (21G 1 ¼) ou 40x12 (18G 1 ½), formando respectivamente, o grupo A (G-A) e grupo B (G-B). No G-A e G-B aspiraram-se 480 e 548 foliculos, respectivamente. Sob lupa estéreo-microscópica avaliou-se os oócitos quanto à qualidade do ooplasma e características das células do cumulus oophorus (grau I, II, III e desnudo). Para análise estatística utilizou-se teste t Student, regressão logistica e teste de proporções, e as diferenças foram consideradas significativas quando P<0,05. A taxa de recuperação entre G-A e G-B não apresentou diferença; 66,5% (330/496)

This study investigated the effects of different concentrations of FSH (10, 50, 100 and 200 ng/ml) in supplemented MEM+ on the development of equine pre-antral follicles that were cultured in vitro for 2 or 6 days. The ovaries (n = 5) from mares in seasonal anoestrus were collected from a local abattoir. Ten ovarian tissue fragments of approximately 3 × 3 × 1 mm were obtained from each animal. The fragments were cultured in situ for 2 days (D2) or 6 days (D6) in MEM+ or MEM+ supplemented with FSH at four different concentrations, establishing the following 11 groups: control (D0); MEM + (D2); MEM + (D6); MEM + 10 ng/ml of FSH (D2); MEM + 10 ng/ml of FSH (D6); MEM + 50 ng/ml of FSH (D2); MEM + 50 ng/ml of FSH (D6); MEM + 100 ng/ml of FSH (D2); MEM + 100 ng/ml of FSH (D6); MEM + 200 ng/ml of FSH (D2); and MEM + 200 ng/ml of FSH (D6). Follicles were observed in only 9.65% (388 of 4,018) of the histological sections. Of the 861 follicles evaluated, 488 were in the primordial stage, and 373 were in various developmental stages; 59.7% were morphologically normal. Regarding the integrity of the pre-antral follicles, the groups with 100 ng/ml FSH of 2-days culture as well as 50, 100 and 200 ng/ml FSH of 6-days culture provided the best results. In conclusion, the in vitro culture of abattoir-derived equine ovarian fragments presented better morphological integrity when supplemented with FSH for 6 days, in comparison with the MEM culture group. However, no clear effects were observed with FSH regarding the promotion of activation from a primordial to a developing follicle.

A placentite é uma das principais causas de abortamentos, natimortos, perda perinatal e dificuldade em conceber na temporada reprodutiva subsequente. O objetivo deste trabalho é revisar os aspectos relacionados com a etiologia, sinais clínicos, métodos diagnósticos e tratamento da placentite em éguas. A placenta equina é fixada ao endométrio através de microcotilédones, com exceção da estrela cervical, sendo esta a região mais afetada pela afecção. Os principais agentes etiológicos da placentite são as bactérias, seguidas dos fungos, vírus e protozoários. A incompetência das barreiras anatômicas é o principal fator predisponente para a contaminação uterina. Os principais sinais clínicos são lactação precoce, corrimento vaginal, morte fetal e abortamento. Uma das formas de diagnostico é feita através da ultrassonografia, avaliando a placenta, fluido uterino e o concepto. Não há tratamento definido, mas o objetivo é combater a infecção, reduzir a inflamação e controlar a atividade do miométrio. O prognóstico tanto para a égua quanto para o potro é variável, dependendo do tempo de gestação, grau de infecção e eficácia do tratamento. É importante monitorar o terço final de gestação das éguas, favorecendo o diagnóstico precoce para obter sucesso com o tratamento e nascimento de um potro saudável.Palavras-chave: Equinos. Abortamento. Morte embrionária. Dismaturo. PLACENTITIS IN MARES: A REVIEW SUMARY:Placentitis is one of the main causes of abortion, stillbirth, perinatal loss and difficulty in conceiving in the subsequent reproductive season. The objective of this work is to review aspects related to etiology, clinical signs, diagnostic methods and treatment of placentitis in mares. The equine placenta is attached to the endometrium through micro-cotyledons, with the exception of the cervical star, and the region is most affected by the condition. The main etiological agent of placentitis is bacteria, followed by fungi, viruses and protozoa. The uterine contamination due to failure of the anatomic barriers is the fundamental predisposing factor. The main clinical signs are early lactation, vaginal discharge, fetal death and miscarriage. One of the forms of diagnosis is made through ultrasonography, evaluating the placenta, uterine fluid and the concept. There is no definite treatment, but the goal is to combat infection, reduce inflammation and control myometrial activity. The prognosis for both mare and foal is variable, depending on the time of gestation, degree of infection and efficacy of the treatment. It is important to monitor the final third of gestation of the mares, favoring the early diagnosis to be successful with the treatment and birth of a healthy foal.

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The antral follicle count (AFC) has been pointed out as one of the main factors that influence the efficiency of reproductive biotechniques. Thus, AFC is considered a fertility marker, which has high repeatability in the same animal. The objective of this study was to evaluate AFC in cows at AI (nonpregnant) and at two moments of gestation (30 and 60 days). For this, 35 pregnant cows, aged 23 to 99 months, confined in a freestall system, kept in the same environmental conditions (24°46'28”S, 49°56'42”W) and under the same feeding conditions, were selected for this study. The same technician performed the AI of the females, after detection of natural or induced oestrus, with 25mg of Dinoprost (Lutalyse) intramuscularly (IM). We established AFC (antral follicles ≥3mm diameter) using a convex intravaginal transducer at the time of AI and at 30 and 60 days of pregnancy. Females were distributed in groups of low (≤18 follicles, n=11), intermediate (≥24 and ≤28 follicles, n=11), or high AFC (≥30 follicles, n=13). Data were analysed using a generalized linear mixed model (Minitab version 18.1), with significance declared at P ≤ 0.05. In the low AFC group, there was an increase in the mean number of antral follicles in relation to gestation, presenting 14.82±1.36 at the time of AI and 23.45±2.31 and 35.18±3.17 at 30 and 60 days of pregnancy, respectively (P&lt;0.0001). Similarly, the intermediate group presented an increase in mean AFC, from 24.64±0.76 at the time of AI and 29.27±3.32 at 30 days to 43.36±5.91 at 60 days of gestation (P=0.001). There was no difference in the high AFC group at different moments, as these presented 41.92±3.50 at the time of AI, 38.31±3.96 at 30 days, and 50.92±5.49 at 60 days of pregnancy. Although mean AFCs differed (P&lt;0.0001) between the low, intermediate, and high AFC groups at time of AI and at 30 days of gestation, there were no differences in AFC between the three groups at 60 days of gestation (P=0.329). In conclusion, low AFC females presented an increase in the number of antral follicles at gestation moments 30 and 60 days from AI. This finding reinforces the strategy of using this category more efficiently for ovum pickup at the beginning of gestation.