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.

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.

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.

The aim of this study was to evaluate the effect of different concentrations of EGF (Epidermal Growth Factor) on in vitro culture of equine preantral follicles. Ovaries (n=5) obtained at a local slaughterhouse from mares at seasonal anestrus were washed in PBS and alcohol 70 and transported in PBS plus penicillin (200 IU/mL) and streptomycin (200 IU/mL). The inner portion of ovary was divided into 11 fragments of 3x3x1 mm. One fragment of each ovary was immediately fixed in Bouin (control group, D0). The remaining 10 fragments were individually cultured in 24- well culture plates containing 1 mL MEM (Gibco BRL, Rockville, MD, USA) (osmolarity 300 mOsm/L, pH 7.2) supplemented with penicillin (100 IU/mL), streptomycin (100 mg/mL), bovine serum albumin (1.25 mg/mL- Gibco BRL, Rockville, MD, USA), ITS (Insulin- 6.25 g/mL, transferrin - 6.25 g/mL, Selenium - 6.25 ng/mL), pyruvate (0.23 mM), glutamine (2 mM) and hypoxanthine (2mM). This medium was referred as MEM +. Culture was performed for 2 or 6 days with medium change every 2 days. Medium was supplemented with different concentrations of EGF (10, 50, 100 and 200 ng/mL). After culture, fragments were fixed in Bouin and processed for histology. Follicles were classified according to the stage of development (primary or developing) and morphology (normal or degenerated). A total of 825 slides containing 3,300 tissue sections were evaluated. The statistical model used was Proportion test (P u003c 0.05). After two days of culture there was a higher proportion of viable follicles at a concentration of 100 ng/mL EGF (87.5%), while MEM had 44.4%; 10 ng/mL had 22.2%; 50 ng/mL had 46,4% and 200 ng/mL had 64.9%. We observed follicular development in all tested concentrations of EGF after two days of culture. EGF at 100 ng/ml provided the best results, with all follicles in development, while MEM had 50%, 10 ng/mL had 75%, 50 ng/mL had 69.2% and 200 ng/mL had 91.7%. After six days of culture, EGF dose did not alter follicular viability. Regarding the proportion of developing follicles after six days, the best results were obtained with EGF at 10 ng/mL and 50 ng/mL with all follicles classified as developing, and 200 ng/mL with 85.7% of developing follicles. Therefore, EGF at 100 ng/mL promoted the best viability at two days of culture, while there was no difference between treatments for this endpoint after six days of culture. Considering follicular development, EGF at 100 ng/mL was the most effective for two days, whereas the doses of 10, 50 and 200 ng/mL were most effective after 6 days of culture to promote development. We conclude that there is a dynamic demand for EGF in in vitro culture of equine preantral follicles.

The aim of this study was to evaluate the effect of FSH addition in the medium for in vitro culture of preantral follicles of Bos indicus females. Ovaries (n=10) were collected at a local slaughterhouse from five adult cycling Bos indicus cows (Nelore) with body condition score from 2.5 to 3.5 (range 0-5). After collection, ovaries were washed in 70% ethanol and PBS (Embriolife®, Vitrocell, Brazil). The surrounding tissue was removed and ovaries were sectioned longitudinally. The ovarian cortex was divided in 3x3x1 mm fragments. One fragment per animal was immediately fixed in Bouin (non-cultured control, D0). The other fragments (n=8) were individually cultured in 24- well culture plates containing 1 ml of minimum essential medium (MEM, Gibco BRL, Rockville, MD, USA; osmolarity 300 mOsm/l, pH 7.2) supplemented (MEM+) with ITS (6.25 mg/ml insulin, 6.25 mg/ml transferrin, and 6.25 ng/ml selenium), 0.23 mM pyruvate, 2 mM glutamine, 2 mM hypoxantina, 1.25 mg/ml bovine serum albumin (BSA Gibco BRL, Rockville, MD, USA), 20 IU/ml penicillin and 200 mg/ml streptomycin. Unspecified reagents were obtained from Sigma (St. Louis, MO, USA). MEM + medium was added to different concentrations (50, 100 and 200 ng/ml) FSH (Folltropin®, Bioniche Canada Inc, Ontario, Canada). The in vitro culture medium was tested during two (D2) or six (D6) days. Every two days, the culture media was replaced by fresh aliquots. For the analysis of integrity and degree of follicular development we used histology with periodic acid-Schiff (PAS) and hematoxylin staining. The classification was based on the evaluation of follicular development stage (primordial, primary and secondary) and morphological integrity as normal or degenerated. Data were submitted to ANOVA (p ≤ 0.05). We evaluated 2,250 preantral follicles (normal or degenerated), being 772 primordial and 1,478 in development. After two days of culture, FSH at 100 ng/ml provided a higher proportion (51.2%; 128/250) of follicles morphologically intact when compared to the other groups: 27.2% (68/250) to MEM; 30.4% (76/250) to 50 ng/ml; 45.2% (113/250) to 200 ng/ml (p 0.05). We conclude that MEM+ supplemented with 100 ng/ml FSH for two or six days of in vitro culture was the most effective treatment to provide development and morphological integrity of preantral follicles from Bos indicus cows.

The aim of this study was to determine the variations in the constituents of the erythrocyte and leukocyte count in the peripartum female sheep of northern Parana. Crossbred ewes (n = 9) underwent venipuncture every 24 h in the five days prior to birth, in the moment of birth, and 12 h, 1, 2, 3, 4, 8 and 16 days postpartum. The samples were collected in EDTA tubes and the following analyzes were performed: Globular Volume (VG), Hemoglobin (Hb), Red Blood Cells (HE), Mean Corpuscular Volume (VCM), Mean Corpuscular Hemoglobin Concentration (CHCM), Mean Corpuscular Hemoglobin (HCM), Red Cell Distribution Width (RDW), and total count and differential leukocyte. The data were analyzed with descriptive statistical analysis and showed that pregnancy and puerperal influence on leukocyte profile of sheep with a predominance of neutrophil next of birth, in the moment of birth and 12 h postpartum. The hematological values showed no significant variations during the experiment.and 12 h postpartum. The hematological values showed no significant variations during the experiment.

The aim of this study was to evaluate the effects of different concentrations of ascorbic acid (25, 50, and 100 μg/mL) in supplemented minimum essential medium (MEM+) on the development of equine preantral follicles that were cultured in vitro for 2 or 6 days. The contralateral ovaries (n = 5) from five mares in seasonal anestrus were collected from a local abattoir. Nine ovarian tissue fragments of approximately 5 × 5 × 1 mm were obtained from each animal. One fragment was immediately fixed and subjected to histologic analysis (control group; Day 0), and the other eight were placed in PBS supplemented with penicillin (200 IU/mL) and streptomycin (200 mg/mL) at 4 °C for 1 hour (during transport to the laboratory). The fragments were cultured in situ for 2 days (D2) or 6 days (D6) in MEM+ or MEM+ plus ascorbic acid at three different concentrations, establishing the following nine groups: control; MEM+ (D2); MEM+ (D6); MEM+ 25 μg/mL of ascorbic acid (D2); MEM+ 25 μg/mL of ascorbic acid (D6); MEM+ 50 μg/mL of ascorbic acid (D2); MEM+ 50 μg/mL of ascorbic acid (D6); MEM+ 100 μg/mL of ascorbic acid (D2); and MEM+ 100 μg/mL of ascorbic acid (D6). The preantral follicles were classified according to their stage (primordial, primary, secondary, or antral) and their morphology (normal or abnormal). Slides (n = 951) including 4450 histologic sections were evaluated. Follicles were observed in only 4.85% (216 of 4450) of the histologic sections. Of the 407 follicles evaluated, 120 were in the primordial stage and 287 were in different developmental stages; additionally, 43.5% were morphologically normal. After 6 days of culture, the groups cultured with 50 and 100 μg/mL of ascorbic acid differed in terms of follicular development compared with the other groups. On the basis of occurrence of follicular development and the presence of viable follicles, it can be concluded that a positive effect of culture for 6 days in MEM+ supplemented with 50 and 100 μg/mL of ascorbic acid was observed on equine ovarian fragments.

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.

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 aim of this study was to evaluate the effect of different concentrations of EGF on in vitro culture of preantral follicles enclosed in equine ovarian fragments. Ovaries (n=5) from mares in seasonal anoestrus were collected from a local slaughterhouse, washed in PBS and ethanol 70% and transported for one hour in PBS plus penicillin (200 IU/mL) and streptomycin (200 mg/mL) at 4°C. At the laboratory, the internal part (parenchyma) of the ovary was divided into 11 fragments of approximately 3x3x1 mm (125-135 mg). One fragment of each ovary was immediately fixed in Bouin (control, D0). The other 10 fragments were individually cultured in culture dishes containing 1mL aliquots of MEM (Gibco BRL, Rockville, MD, USA) (osmolarity 300 mOsm/L, pH 7.2) supplemented with penicillin (100 IU/mL) streptomycin (100 mg/mL), bovine serum albumin (1.25 mg/mL- Gibco BRL, Rockville, MD, USA), ITS (insulin - 6.25 µg/mL transferrin - 6.25 µg/mL selenium - 6.25 ng/mL), pyruvate (0.23 mM), glutamine (2 mM) and hypoxanthine (2 mM), which is referred as MEM+. Culture was performed at 39°C in an atmosphere with 5% CO2 in air and saturated humidity for 2 days. Medium was supplemented with different concentrations of EGF (10, 50, 100 and 200 ng/mL). 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, and according to their morphology as normal or degenerated. Data were compared by nonparametric analysis of variance (ANOVA, P<0.05). A total of 450 slides containing 1800 sections were analyzed including the control group and treatment groups. Overall, 198 follicles were identified, of which 93 (47%) were primordial, 105 (53%) were developing and 58% were classified as morphologically normal. Follicular development was observed in all tested concentrations of EGF (MEM - 75% (3/4), 10ng/mL - 100% (4/4), 50ng/mL - 85,7% (12/14), 100ng/mL - 100% (6/6) e 200ng/mL - 96% (24/25)). We concluded that MEM+ medium supplemented with these concentrations of EGF allowed development of follicles in equine ovarian fragments cultured for two days.

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.