A prestacao de servicos pelo Laboratorio de Biotecnologia da Reproducao Animal (REPROA) da Universidade Estadual de Londrina (UEL) constitui uma atividade extensionista de grande importância aos produtores. Assim, o presente trabalho visa relatar a casuistica e os beneficios do auxilio tecnico-veterinario nas propriedades agropecuarias e criatorios do norte paranaense no periodo de 30/08/2016 a 30/08/2019. Um total de 1494 animais foram atendidos neste periodo, sendo 53,8% bovinos, 28,2% ovinos, 17,6% equinos e 0,4% de caprinos e suinos. Os atendimentos foram realizados no REPROA-UEL ou em propriedades conforme a demanda. Nos atendimentos solicitados foi contemplado em bovinos – 9 partos eutocicos, 4 auxilios ao parto distocico, 2 casos de retencao placentaria, 98 inseminacoes artificiais, 169 diagnosticos de gestacao, 1 caso de vaginite, 219 exames andrologicos, 3 endometrites, 26 sexagens, 30 protocolos de IATF e 243 exames ginecologicos; equinos – 62 exames ginecologicos, 2 sexagens, 4 partos eutocicos, 1 auxilio ao parto distocico, 59 acompanhamentos foliculares, 4 colheitas de embriao, 5 exames andrologicos, 11 inseminacoes artificiais, 3 endometrites, 1 caso de cistite hemorragica idiopatica, 1 caso de colica espasmotica por superdosagem de PG2a, 1 cisto endometrial e 109 diagnosticos de gestacao; ovinos – 345 diagnosticos de gestacao, 54 exames ginecologicos, 21 exames andrologicos e 2 prolapsos cervico-vaginais; caprinos – 2 partos distocicos e 1 exame andrologico; e suinos - 2 partos distocicos. O atendimento especializado proporcionou resultados positivos a um elevado numero de animais, evidenciando que o projeto se faz necessario e viavel, representando um importante ponto de apoio a assistencia reprodutiva especializada para regiao.

The effect of different concentrations of alpha lipoic acid (ALA) on the development and morphology of preantral follicles, as well as the proliferative activity of granulosa cells, was assessed after short-term culture. Ovaries (n = 5) of five seasonal anestrous mares were harvested in a local abattoir. At the laboratory, nine ovarian fragments (5 × 5 × 1 mm) from each animal were used. One fragment was immediately fixed in Bouin and subjected to histological and immunohistochemistry (proliferating cell nuclear antigen, PCNA) analyses (noncultured group; D0 = day 0). The other eight fragments were cultured in situ for two (D2) or six (D6) days in MEM+ or MEM+ plus ALA (50, 100, or 250 μM). After culture, fragments were subjected to histology and PCNA analyses. After two days of culture, ALA 50 and ALA 100 had the greatest (P < 0.05) percentage of normal primordial follicles (97.2 and 95.1%, respectively), when compared to other groups, and did not differ (P > 0.05) from the fresh noncultured control group. Furthermore, the total percentage of normal follicles was greater (P < 0.05) in the ALA 50 and ALA 100 than in the MEM-D2 group. After six days of culture, the highest (P < 0.05) proliferative activity of granulosa cells in developing follicles was observed for the groups MEM+ (92.9%), ALA 50 (100%), and ALA 100 (96.4%). In conclusion, the results of this study demonstrated that (1) ALA 50 and ALA 100 preserved the morphological integrity of equine primordial follicles for up two days of culture, and (2) granulosa cells of developing follicles enclosed in ovarian tissue and cultured for up to six days in MEM+ with or without ALA were highly stained by PCNA.

Oocyte mitochondria are increased in number, smaller, and rounder in appearance than mitochondria in somatic cells. Moreover, mitochondrial numbers and activity are narrowly tied with oocyte quality because of the key role of mitochondria to oocyte maturation. During oocyte maturation, mitochondria display great mobility and cluster at specific sites to meet the high energy demand. Conversely, oocyte mitochondria are not required during early oogenesis as coupling with granulosa cells is sufficient to support gamete's needs. In part, this might be explained by the importance of protecting mitochondria from oxidative damage that result in mutations in mitochondrial DNA (mtDNA). Considering mitochondria are transmitted exclusively by the mother, oocytes with mtDNA mutations may lead to diseases in offspring. Thus, to counterbalance mutation expansion, the oocyte has developed specific mechanisms to filter out deleterious mtDNA molecules. Herein, we discuss the role of mitochondria on oocyte developmental potential and recent evidence supporting a purifying filter against deleterious mtDNA mutations in oocytes.

The aim of this work was to investigate the cell proliferative activity of multi-oocyte follicles in adult sheep. Ovaries (n = 64) were processed for histology and immunostaining with proliferation cell nuclear antigen (PCNA). Multi-oocyte follicles were observed in 87.50% (28/32) pairs of ovaries. Positive immunostaining for anti-PCNA antigen was observed in 97.8% of the one-oocyte follicles and 97.2% of multi-oocytes follicles (p > 0.05). Oocytes included in the same follicle showed a similar pattern of PCNA staining and appear to be in the same developmental stage. (C) 2016 Elsevier B.V. All rights reserved.

Mitochondria are known to play a major role in providing oocytes with developmental competence, but the mechanisms underlying this function are elusive. Compared to other cell types in an organism, the oocyte has the greatest amount of mitochondrial DNA (mtDNA) and mitochondria that distinguish by their small size and rounded shape. These unique characteristics are dependent on events of mitochondrial fusion and fission, which are determinant to organelle function and inheritance. Thus, to address the role of mitochondrial fusion in oocytes, the genes Mfn1 and Mfn2 were conditionally knocked out by Zp3-directed expression of Cre recombinase. Towards this aim, a heteroplasmic mouse lineage containing ~50% of mutant mtDNA (NZB) was used. Wild-type (WT) or knockout oocytes for Mfn1 (Mfn1-null), Mfn2 (Mfn2-null) or both (Mfn-dm) were compared regarding their developmental competence, mitochondrial function and inheritance of NZB mtDNA. Data were evaluated by oneway ANOVA followed by Duncan posthoc test. Differences with P < 0.05 were considered significant. Mating of females containing Mfn1-null oocytes with WT males did not result in birth. Infertility in this case was associated with a failure in ovulation, accumulation of secondary follicles (251 ± 36 vs. 54 ± 12 follicles) and arrest of meiotic progression when oocytes were in vitro cultured. Mfn1-null oocytes also presented smaller diameter (57.8 ± 0.73 vs. 85.7 ± 0.49 μm) and lower levels of mtDNA (39,263 ± 2,878 vs. 198,164 ± 18,611 copies), ATP (0.03 ± 0.016 vs. 0.46 ± 0.076 pmol) and mitochondrial membrane potential (∆Ψm; 0.71 ± 0.03 vs. 1.00 ± 0.03 a.u.). The lower abundance of Alix protein in Mfn1-null oocytes, a known marker of extracellular vesicles, suggests that secretion of oocyte-derived paracrine factors was deficient. In regards to Mfn2-null oocytes, in spite of the smaller diameter (78.6 ± 0.68 μm) and lower ovulation rate (10.3 ± 4.29 vs. 23.0 ± 3.82 oocytes), these oocytes were not affected based on the level of mtDNA, ATP and ∆Ψm. As a result, the number of offspring born to Mfn2-null and WT oocytes did not differ. Moreover, the mice born to Mfn2-null oocytes grew normally, but had higher levels of blood glucose, both baseline (135 ± 3.9 vs. 115 ± 3.5 mg/dl) and fasted (172 ± 7.2 vs. 137 ± 4.1 mg/dl). Surprisingly, Mfndm oocytes presented a mild defect when compared to WT ones, characterized by slightly smaller diameter (76.7 ± 0.79 μm), intermediate level of mtDNA (104,215 ± 12,657 copies) and ∆Ψm (0.81 ± 0.02 a.u.), and normal level of ATP, ADP and ATP/ADP. However, Mfn-dm oocytes contained smaller and fewer mitochondria, which aggregated with the endoplasmic reticulum. In spite of being ovulated, Mfn-dm showed a disrupted meiotic spindle and an incompetence to develop to term. In respect to the mitochondrial inheritance, the level of NZB mtDNA decreased in WT (-10.1 ± 1.25) and Mfn1-null (-10.3 ± 1.58) oocytes in comparison to their donor females, suggesting a selection in the germline against mutant mtDNA. However, this selection was less effective in Mfn2-null (-3.0 ± 1.50) and Mfn-dm (-4.4 ± 1.21) oocytes. In summary, this work gives evidence that Mfn1 is essential to provide oocytes with developmental competence whereas Mfn2 is involved in the elimination of mutant mtDNA. These findings support a key role of mitofusins in adapting mitochondria to meet oocyte’s needs and prevent inheritance of mutant mtDNA.

The diameter of follicles from which oocytes are retrieved and the time of the first cleavage potentially influence competence and epigenetic reprogramming in early embryonic development. This study aimed to investigate the developmental potential and the occurrence of monomethylation at lysine 4 of histone H3 (H3K4me) in bovine blastocysts of early, intermediate and late cleavage after fertilizing oocytes from small and large follicles. Small (≤ 2 mm) and large follicles (4-8 mm) from slaughterhouse ovaries (n = 1982) were punched. Among the collected oocytes, 699 from ≤ 2 mm diameter follicles and 639 from 4-8 mm diameter follicles were subjected to in vitro maturation and fertilization. The presumptive zygotes were cultured, and cleavage rates were evaluated by separating the embryos into early (≤ 28 h post-IVF), intermediate (u003e 28 h and ≤ 34 h post-IVF) and late (u003e 34 h and ≤ 54 h post-IVF) groups. The blastocyst rates were further evaluated after 7 and 8 days of culture. The blastocyst and cleavage rates were compared by logistic regression and differences were considered statistically significant at a confidence level of 95% (P u003c 0.05). Among blastocysts, we randomly selected 5 embryos per group and investigated H3K4me by immunofluorescence. The percentage of late-cleaved embryos was higher (P u003c 0.05) than that of earlycleaved embryos for 4-8 mm follicles (late: 30% vs. early: 19%) and ≤ 2 mm follicles (late: 33.8% vs. early: 16.6%), indicating that most embryos start the first cell division cycle later. The blastocyst rate for the 4-8 mm group (36.3%) was higher than that for the ≤ 2 mm group (22.9%, P u003c 0.05). In addition, the blastocyst rates for the early and intermediate cleavage groups (45.3% and 33.8%, respectively) were higher than that for the late cleavage group (13.5%, P u003c 0.05). The blastocysts from all the groups displayed H3K4me staining by immunofluoresence; the staining was particularly intense in the trophectoderm region and was weak or absent in the inner cell mass region. Data from this study demonstrate that higher blastocyst embryo rates are obtained from embryos that cleave within 34 hours after fertilization and from those produced from follicles of 4 to 8 millimeters in diameter, indicating a greater ability of these embryos to develop to the stage of embryonic preimplantation. Furthermore, the presence of monomethylation at H3K4 in all the evaluated blastocysts suggests that this histone modification plays a key role in maintaining embryo viability at this important developmental stage.

The aim of this study was to evaluate the effects of different concentrations of GDF-9 on in vitro culture of equine ovarian fragments. Ovaries (n=5) from mares in the anovulatory season were collected at local slaughterhouse and washed in 70o ethanol and PBS, and transported for one hour in PBS plus penicillin (200 IU/mL) and streptomycin (200 mg/mL) at 4oC to the laboratory, where the internal portion of the ovary was divided into 9 pieces of approximately 5 x 5 x 1mm. One fragment of each ovary was immediately fixed in Bouin (control group, D0). The other 8 fragments were cultured individually in culture dishes containing 1mL of MEM (MEM control) or base medium supplemented with GDF-9 (0, 50, 100 or 200 μg/mL). MEM+ was MEM (Gibco BRL, Rockville, MD, USA – 300 mOsm/L osmolarity, pH 7.2) supplemented with pyruvate (500 μL), glutamine (500 μL), hypoxanthine (500 μL), ITS, BSA (1.25 mg/mL-Gibco BRL, Rockville, MD, USA), penicillin (500 μL) and streptomycin (500 µL). Culture was performed at 39°C in an atmosphere with 5% CO2 in air and saturated humidity for 2 or 6 days. Medium was replaced every 2 days. After culture, fragments were fixed in Bouin and processed for histology. Follicles were classified according to their stage of development as primordial, primary, secondary and antral follicles, and according to their morphology as normal or degenerated. Data were compared by nonparametric analysis of variance (Kruskal Wallis test), considering significant differences with P<5%. Overall, 675 slides were evaluated with an average of 4 ovarian fragments in each. We found 552 follicles, of which 68 (12.3%) were developing follicles in the control group (MEM+), 355 (64.3%) were primordial, 197 (35.7%) were developing and 379 (68.6%) were morphologically normal. Fragments cultured with 50 μg/mL of GDF-9 for two days of had higher proportion of developing follicles (9%; 50/552). We concluded that MEM supplemented with 50 µg/mL of GDF-9 provided better follicle development during in vitro culture of equine ovarian fragments for two days.

Multi-oocyte follicles (MOFs) have been often described in studies of follicular population. However, their development and proliferative activity are poorly studied. The purpose of this study was to determine the frequency and activity of multi-oocytes follicles in ovine ovaries. Pairs of ovaries (n = 32) obtained from a slaughterhouse were evaluated for cell proliferative activity through the expression of Proliferation Cell Nuclear Antigen (PCNA) and the protein detected by the antibody Ki-67. The analysis for CPNA was performed by histological score and due to the absence of normality and homoscedasticity, data were analyzed using the Wilcoxon test for dependent samples. There was immunostaining for PCNA in all stages of follicular development and there was no difference (p > 0.05) between the immunoblots of oocytes from follicles containing one oocyte and multi-oocytes follicles. Multi-oocytes follicles were found in greater amounts in the left ovary (p = 0.03). There was no positive immunostaining for Ki-67 in primordial follicles. However, in all other development stages, Ki-67 immunostaining was observed in follicles with one oocyte and MOFs. Thus, MOFs in sheep ovaries showed proliferative activity and are possibly able to develop to antral stages.

The investigation of morphological characteristics of the population of preantral follicles is important for the understanding of physiological processes such as folliculogenesis and for new reproductive strategies. The aim of this study was to morphologically characterize the population of sheep ovarian follicular reserve. Ovine ovaries (n = 32) were histologically processed and analyzed by light microscopy. The follicles and oocytes showed the following average diameters: (primordial with one oocyte) 30.96 ± 4.6 m and 25.09 ± 1.61 mm; (multioocyte primordial) 48.62 ± 5.66 m and 24.31 ± 3.35 mm; (primary) 50.96 ± 8.52 m and 34.42 ± 4.50 mm; (secondary) 86.48 ± 21.85 m and 49.01 ± 11.49 mm; (antral) 226.26 ± 69.93 m and 66.26 ± 11.01 micrometers. Data were compared were using the Wilcoxon test for dependent samples. There was no difference between oocyte diameter in primordial follicles with one or more oocytes. Multioocyte follicles at the primordial stage were observed in greater amounts than secondary follicles, and their prevalence was similar in the right and left ovaries (p = 0.792). Regarding the macroscopic analysis, data were subjected to Spearman correlation test. There was a positive correlation between follicular population and ovarian diameter (p < 0.01), ovarian weight (p < 0.05) and ovarian volume (p < 0.05). The was not correlated with the other variables. However, correlation of total number of multioocyte follicles with follicular average population approached significance (p = 0.056). These results suggest that multioocyte primordial follicles have morphology and developmental capacity similar to primordial follicles with one oocyte.

The aim of this study was to evaluate the effect of adding different concentrations of ascorbic acid to in vitro culture of equine preantral follicles. Ovaries (n = 5) from mares in the anovulatory season were collected at a local slaughterhouse and washed in 70% ethanol and PBS. The internal region (parenchymal) was divided into 9 pieces of approximately 5x5x1 mm. One fragment was immediately fixed in Bouin (control) and the other 8 pieces were transported for one hour to the laboratory in PBS plus penicillin (200 IU/mL) and streptomycin (200 mg/mL) at 4°C. At the laboratory, 8 fragments were individually cultured in culture dishes at 39°C in an atmosphere with 5% CO2 in air with saturated humidity for two (D2) or six (D6) days in 1 ml of minimum essential medium (MEM) - (Gibco BRL, Rockville, MD, USA) (osmolarity 300 mOsm/L pH 7.2) supplemented with ITS (6.25 ng/mL insulin, 6.25 ng/mL transferrin, 6 25 ng/mL selenium), 0.23 mM pyruvate, 2 mM glutamine, 2 mM hypoxanthine, 1.25 mg/mL bovine serum albumin, 200 IU/mL penicillin and 200 mg/mL streptomycin (MEM+). The treatments consisted of MEM+ supplemented with different concentrations of ascorbic acid (25, 50, or 100 µg/ml). Medium was replaced every two days. After culture, ovarian fragments were fixed in Bouin and processed for histology. Follicles were classified according to their stage of development in primordial, primary, secondary and antral follicles, and according to their morphology in normal or degenerated. Data were compared by nonparametric analysis of variance (Kruskal Wallis test), considering 5% significance level. Overall, 951 slides were evaluated containing 4,450 sections. Follicles were present in 4,85% (216/4450) of the evaluated histological sections. We classified 43.5% of the follicles as morphologically normal. The control group showed a higher proportion of primary (89 %) than developing follicles (11 %). Regarding follicular development, fragments treated with MEM (D6) and 25 µg/ml of ascorbic acid (AA 25D6) presented a higher percentage of developing follicles in comparison with primordial follicles. This group showed greater follicular development (26.3% of primordial follicles and 73.7% of developing follicles) compared to the other groups analyzed (P ≤ 0.05). We conclude that ascorbic acid at 25 µg/ml was effective to promote the development of equine primordial follicles for six days of culture.

Genes associated to organogenesis, morphogenesis and embryonic development are temporarily suppressed in pluripotent cells of mammalian embryos by the trimethylation of lysine 27 of histone H3 (H3K27 me3). H3K27 methylation is catalyzed by enzymes of the Polycomb Repressive Complex 2 (PRC2); however, this process is not entirely known. The AdoHcy hydrolase inhibitor 3-Deazaneplanocin A (DZNep) can prevent the action of the PRC2 enzymes, therefore inhibiting H3K27 me3. Nevertheless, there are no reports of the action of DZNep in embryos. The aim of this study was to evaluate the blastocyst rate and the cell number of bovine embryos cultured with 5 µM DZNep from day 3 (D3) or day 5 (D5) of embryonic development. Oocytes from Nelore females obtained from slaughterhouse ovaries were subjected to in vitro maturation (IVM) for 24 h at 38.5 °C, with 5% CO2 in air and saturated humidity. In vitro fertilization (IVF) was performed with a previously tested frozen-thawed semen from a Nelore bull. Oocytes and spermatozoa remained in coculture for 22 h under the same conditions of IVM. At D3 (considering the day of IVF as D0), the cleaved embryos were randomly separated into 3 groups: exposed to 5 mM DZNep from D3 (DZNep D3; n = 80), exposed to 5 mM DZNep from D5 (DZNep D5; n = 81) or without the addition of DZNep (control group; n = 82). Four replicates were performed, and the blastocyst rates were assessed at D8. All fertilized structures (not only the blastocysts) were fixed in 4% paraformaldehyde and the cell nuclei were stained with DAPI to estimate the number of cells. Blastocyst rates were compared between the treatments using a logistic regression test and the average number of cells by analysis of variance followed by Tukey test, with 5% of significance level. The blastocyst rate obtained with the control group was 34.1%, higher than the ones obtained with groups DZNep D3 (11.2%) and DZNep D5 (7.4%), which did not differ. The average cell number of the control group was 38.7, higher than the average cell number of groups DZNep D3 (7.5) and DZNep D5 (6.4), which did not differ. Based on these results, it can be concluded that H3K27 me3 plays an important role in embryonic gene regulation because its inhibition results in decreased blastocyst rates and the produced embryos have fewer cells.

The objective of this study was to evaluate by immunohistochemistry cellular proliferation in multioocyte follicles from bovine adult ovaries. Ovaries (n=12) from Nelore heifers obtained at a slaughterhouse were fixed in Bouin and submitted to histological processing. Slides were prepared in duplicate (PAS staining-Schiff and Immunohistochemistry) with sections of 5 µm. Screening for multioocyte follicles was performed on PAS stained slides. All multioocyte follicles were submitted to immunohistochemistry with anti-PCNA (Clone PC1O, ZYMED® Laboratories). The level of PCNA-positive cell proliferation was assessed in granulosa cells (score ranging from one to four) and oocytes (qualitative assessment). Data were evaluated only for qualitative aspects and therefore will not be expressed numerically. Data were subjected to ANOVA (p ≤ 0.05). From 12 ovaries examined, we found 12 multioocyte follicles, containing from two to three oocytes. These structures were found predominantly in primordial follicles (n = 10) and primary follicles. There was no difference (p > 0.05) in cell proliferation of granulosa cells from primordial and primary follicles. However, there was a trend of increase in mitotic activity of granulosa cells from follicles multioocyte compared to other follicles. For the mitotic activity in oocytes, PCNA immunostaining was positive in follicles at different stages of development. Immunostaining was stronger in oocytes from multioocyte follicles (p < 0.05), reinforcing the proposition that multioocyte follicles are in constant proliferation. Based on these results, we concluded that adult Bos taurus indicus cattle present multioocyte follicles in early stages of development. The oocytes enclosed in multioocyte follicles do not display the same pattern of PCNA immunostaining, suggesting they are in different stages of development and/or there is some kind of competition or dominance between them.

Mules are generally ignored in the reproductive context due to the classic concept of infertility and absence of ovarian activity. Therefore, currently few studies are conducted on this topic. The aim of this study was investigate the ciclicity in ovaries of mules and report the birth of an asinine foal from a mule. Ovaries (n= 72) from mules were obtained at a local slaughterhouse, ovarian capsules were removed with scissors and scalpel blade. Ovaries were measured and externally inspected for presence of corpora lutea and follicles. The visualized follicles were aspirated with a 20 mL syringe containing heparin solution and needle 40x12 (18G1 ½) for oocyte retrieval. It was possible to verify that 45.8% (33/72) of the ovaries presented corpora lutea and 15.3% (11/72) had follicles > 10mm. It was possible to obtain an oocyte of all follicles aspirated. From all oocytes (n= 11) recovered, three were intact with expanded cumulus cells, while all the others were atretic. Considering the presence of follicles, corpora lutea and expanded cumulus oocytes in the ovaries of mules, we consider the use of this category as recipient for asinine embryos. After identifying regular cycles in a mule, the follicular control was performed after detection of estrus of the donor donkey. When the donkey had a 38 mm follicle and uterine edema grade 3 (scale 1-3), the ovulation was induced with human chorionic gonadotropin (hCG) (Vetecor®, Hertape-Calier, Spain) (2500 IU) IM. On the following day, the donor was covered by a donkey male. The ovulation occurred within 48 hours, detected by ultrasonography. At D8 just five days post ovulation the embryo was collected by uterine flushing via transcervical with a double system with 1 L of lactated ringer's solution. After identification, the embryo was washed in 10 drops Holding plus (Embriolife®, Vitrocell, Brazil) and transferred to the recipient mule, which had signs of estrus two days later after donor. The recipient had ovulation induced (hCG 2500 IU IM) after the detection of 37 mm follicle and uterine edema grade 3. Ovulation was confirmed by ultrasonography. On the day of transfer (D5), the mule had a rigid uterine tone and corpus luteum of approximately 30 mm. The embryo transfer was made at uterine body with transrectal palpation, due to the difficulty for passing the cervix because of its small diameter. After five days the pregnancy diagnosis was performed, with confirmation at 60 days post-transfer, both by transrectal ultrasonography. The eutocic birth of the foal donkey occurred after 372 days gestation. We highlighted that the occurrence of regular cycles in mules may be more frequent than commonly expected. Furthermore, the use of this cyclicality can be considered for use in reproductive biotechnologies such as embryo transfer.