Ethanol, Carnoy, and paraformaldehyde as fixative solutions for histological evaluation of preantral follicles in equine ovarian tissue.

The most adequate fixative solution for equine ovarian tissue is still to be determined as a tool to evaluate the improvement of methodological studies in assisted reproductive techniques and fertility preservation. This study aimed to evaluate a short-time ethanol 70% (ST-EtOH, 45 min) exposure as an alternative fixative compared with two classically fixatives [Carnoy's (CAR) solution and paraformaldehyde 4% (PFA)] at different fixation times (6 h, 12 h). The end points evaluated were morphology and classes of preantral follicles, follicular and stromal cell densities, and follicular and oocyte nuclear diameters in equine ovarian tissue. Ovaries (n = 6) from ovariectomized young mares were fragmented (3 × 3 × 1 mm; 20 fragments/ovary) and fixed in the tested treatments. Overall, a total of 11,661 preantral follicles were evaluated in 1444 histological slides. The ST-EtOH similarly preserved the preantral follicle morphometry and stromal cell density compared to the PFA fixative, regardless of the exposure time. Nonetheless, the CAR fixative solution had the greatest percentage of normal preantral follicles and the highest stromal cell density among all treatments. In conclusion, Carnoy's solution must be preferred compared with ST-EtOH and PFA fixatives for studies concerning the cellular morphology of equine ovarian tissue. Moreover, ST-EtOH fixative is a good alternative for equine ovarian tissue when a quick histological evaluation is required instead of more time-consuming and expensive techniques. Additional studies concerning the impact of different fixatives on the ultrastructure of cellular populations and their compatibility with IHC and molecular techniques in equine ovarian tissue are warranted.

Follicle Selection in Mares as a Model for Illustrating the Many Hormonal and Biochemical Interactions That Drive a Single Physiological Mechanism.

The mechanism for selection of the future dominant or ovulatory follicle in mares involves a relatively abrupt separation in growth rates between the future dominant follicle and several subordinate follicles and is termed diameter deviation. The event is used to illustrate that a coordinated complex of many follicular, hormonal, and biochemical factors interact and interbalance during a single physiological mechanism. For example, a positive effect of follicle stimulating hormone (FSH) on development of all follicles during the growing phase can later involve a positive effect of luteinizing hormone (LH) but apparently only on the future dominant follicle. In turn, the developing and future dominant follicle produces estradiol which at appropriate times and degrees reduces FSH concentrations to accommodate follicle functions at certain levels of FSH. Meanwhile, the estradiol prevents LH from increasing from a useful to an adverse concentration. These interactions enmesh with the production and roles of other factors (e.g., inhibin, insulin-like growth factor) during follicle selection. The wide array of morphological, hormonal, and biochemical activities occur in harmony even when in the same tissue and often at the same time.

High-definition ultrasonography in the evaluation of the reproductive tract of bitches during the follicular phase of the estrous cycle.

The aim was to sonographically evaluate the reproductive tract of bitches during the follicular phase of the estrous cycle using High Density (HD) ultrasonic techniques. Females (n = 8) were evaluated at five different times throughout the follicular phase, as determined by vaginal cytology and blood progesterone concentrations. Ultrasonic exams were performed using the ACUSON S2000/SIEMENS device utilizing a multifrequency HD transducer (5.5-18 MHz). Videos of the ovaries were obtained and recordings were evaluated using a DICOM viewer software for counting and measuring the ovarian structures, which were assigned to groups based on diameter in mm: G1: ≤ 1; G2: from 1.01 to 3.5; G3 from 3.51 to 5.5; G4: from 5.51 to 10. There was a greater uterine thickness with the progression of the follicular phase (P < 0.05). Six distinct regions were identified in the uterine wall. The ovarian dimensions increased (P < 0.05) as stage of the follicular phase advanced. There was fluid detected around the ovaries after ovulation. There was a characteristic fat tissue hyperechogenicity around the ovaries at all timepoints. There was a difference in the number of ovarian structures of each dimension group at each time there were assessments (P < 0.05). There was a difference in diameter of the largest ovarian structure and in average value of wall thickness at all timepoints when there were evaluations (P < 0.05). The HD ultrasonography technique provides for excellent image resolution, allowing for a more precise characterization of the bitch's reproductive structures and changes occurring during the follicular phase of the estrous cycle.

Factors influencing pre-ovulatory follicle diameter and weaning-to-ovulation interval in spontaneously ovulating sows in tropical environment.

Follicle development and timing of ovulation are indicators of the reproductive performance of sows. The present study aimed to determine factors influencing pre-ovulatory follicle diameter and weaning-to-ovulation interval (WOI) in spontaneously ovulating sows in tropical climates with special emphasis on breed, parity and backfat thickness at weaning. In total, 80 sows were included in the study. Follicle development was determined by using transrectal real-time B-mode ultrasonography every 6 hr after standing oestrus. Weaning-to-oestrous interval (WEI), oestrous-to-ovulation interval (EOI), WOI and the diameter of graafian follicles were investigated in relation to breed, parity number (1, 2-3 and 4-7) and backfat thickness (low, moderate and high) of sows. Overall, WEI, EOI, WOI and the pre-ovulatory follicle diameter were 92.5 ± 21.6 hr, 64.3 ± 19.3 hr, 156.3 ± 29.1 hr and 10.3 ± 2.0 mm, respectively. Pre-ovulatory follicle size was smaller in primiparous sows compared with sows of greater parity, 4-7 (9.7 ± 0.51 and 11.7 ± 0.52 mm, respectively, p < .05). Weaning-to-ovulation interval was positively correlated with WEI (r = 0.75, p < .001) and EOI (r = 0.66, p < .001), but negatively correlated with size of the graafian follicle (r = -0.34, p < .01). Sows with a shorter WEI had a larger pre-ovulatory follicle diameter (at 64 hr after oestrus) (r = -0.37, p < .01). Sows with low backfat thickness had a WOI 23.4 hr longer than those with moderate backfat thickness (p < .05) and 17.6 hr longer than sows with a high backfat thickness (p = .140). The follicle diameter in primiparous sows with high backfat thickness (11.7 ± 1.1 mm) was higher than in those with low (8.9 ± 0.7 mm, p < .05) or moderate (8.6 ± 0.8, p < .05) backfat thickness. In conclusion, factors influencing follicle diameter and WOI in sows included parity number and backfat thickness at weaning. The impact of backfat thickness on follicle diameter, WEI and WOI was most pronounced in primiparous sows.

Does a Large (>24 mm) Follicle Yield a Competent Oocyte/Embryo?

AIM: To evaluate the effect of large follicular size (≥24 mm) at day of oocyte retrieval on oocyte/embryo quality. PATIENTS AND METHODS: A cohort study was conducted in a single tertiary medical center between July 2018 and May 2019. Before ultrasound-guided follicular aspiration, follicles were measured and divided into 2 groups according to their maximal dimensional size: large: ≥24 mm and normal: <24 mm. Microscopic examination of the follicular aspirates was performed by an embryologist. Each follicle aspirated was evaluated for oocyte maturation, oocyte fertilization, and embryo quality. RESULTS: 428 follicles were measured, including 383 (62.81%) in the normal and 45 (14.06%) in the large follicle groups. Oocytes were achieved during aspiration from 297 (75.5%) and 29 (64.4%) of the normal and large follicle groups, respectively (p = 0.05). No in-between group differences were observed in mature oocyte (MII), fertilization, and top-quality embryo (TQE) rates. Nevertheless, once a zygote (2PN) was achieved, a trend toward a higher TQE rate/2PN was found in the large follicle group (16/19 [84.2%] vs. 115/171 [67.3%]; p = 0.062). CONCLUSION: While a nonsignificant decrease in oocyte recovery rate was found in follicles ≥24 mm, the zygote and TQE per follicle were comparable.

Re-evaluation of reproductive cycle and fecundity of finetooth sharks Carcharhinus isodon (Valenciennes 1839) from the Northwest Atlantic Ocean, with new observations on ovarian cycle and reproductive endocrinology of biennially reproducing sharks.

To provide updated information on life history for improved fishery management, the reproductive cycle of the finetooth shark Carcharhinus isodon from Northwest Atlantic (NWA) populations was investigated by examining temporal changes in morphology and histology of reproductive organs. Changes in plasma concentrations of gonadal sex hormones in relation to reproductive stage were also examined. Increases in testis width, epididymis head width, plasma testosterone concentrations and occurrence of mature spermatozoa were observed in male sharks between December and April, suggesting a seasonal pattern in reproduction that culminates with copulatory activity in May. Increases in maximum follicular diameter, oviducal gland width, plasma 17ß-estradiol concentrations and occurrence of vitellogenic follicles were observed in non-pregnant female sharks during the same time period along with the occurrence of newly pregnant females in May, demonstrating strong synchronicity between male and female reproductive cycles. Pregnant females bearing full-term embryos were also observed in May, indicating that parturition occurs between mid-May and early June and gestation requires 12 months. Only transient temporal changes in follicle size and oviducal gland width were observed in pregnant females, indicating that reproductive periodicity is biennial; nonetheless, a single female exhibiting signs of concurrent vitellogenesis and pregnancy was observed. Mean brood size ± S.D. was 3.9 ± 0.9 offspring/female. Fecundity was not significantly correlated with female size, in part due to an unexpectedly high rate of early embryo mortality, which occurred in 11% of pregnant females, and was more common in larger individuals. Changes in ovarian activity during mid-pregnancy were observed, suggesting possible roles for the ovary in regulating some aspects of early to mid-gestation. This study confirms that earlier characterizations of the reproductive cycle and fecundity in NWA finetooth sharks remain valid for use in fishery management. This study also highlights unusual features of finetooth shark pregnancy (e.g., early embryo death, mid-pregnancy changes in ovarian function) that may have broader relevance to understanding elasmobranch reproduction.

Reproductive cycle and fecundity of the bonnethead Sphyrna tiburo L. from the northwest Atlantic Ocean.

The present study examined temporal changes in plasma sex hormone concentrations and the morphology and histology of reproductive organs in mature northwest Atlantic (NWA) bonnetheads Sphyrna tiburo L. to characterize reproductive cycle, breeding periodicity and fertility in this still poorly studied population. Progressive increases in testis width, epididymis head width, plasma testosterone (T) concentrations, and occurrence of mature spermatozoa were observed in male S. tiburo from June to September, demonstrating that spermatogenesis occurs during the summer. Nonetheless, increases in maximum follicle diameter, oviducal gland width, plasma 17ß-estradiol and T concentrations, and occurrence of vitellogenic follicles were not observed in mature females until between October and April, demonstrating non-synchronous patterns of gametogenesis in males and females. Fresh copulatory wounds were observed in females collected during late September along with histological evidence for sperm presence in the oviducal gland between September and April, confirming a 6- to 7 month period of female sperm storage. Ovulation occurred between mid-April and early May in concert with increases in female plasma progesterone concentrations. Gestation occurred during a 4.5- to 5 month period between May and early September, and 97% of mature females collected during this period were gravid, indicating a highly synchronized, annual reproductive periodicity. Brood size was significantly correlated with maternal size and ranged from 1 to 13 pups with a mean ± S.D. of 8.1 ± 2.2, which was significantly lower than reported in Gulf of Mexico (GOM) populations. The occurrence of non-fertile offspring was observed in 17% of broods with a range of 1-7 non-fertile eggs present in individual females. Thus, as previously reported in GOM S. tiburo, this unusual form of infertility also appears to be prevalent in the NWA population and requires further study. This study has demonstrated meaningful differences in reproductive biology of these populations, emphasizing the need for region-specific approaches for population management.

CR-Unet-Based Ultrasonic Follicle Monitoring to Reduce Diameter Variability and Generate Area Automatically as a Novel Biomarker for Follicular Maturity.

Follicle size is closely related to ovarian function and is an important biomarker in transvaginal ultrasound examinations for assessing follicular maturity during an assisted reproduction cycle. However, manual measurement is time consuming and subject to high inter- and intra- observer variability. Based on the deep learning model CR-Unet described in our previous study, the aim of our present study was to investigate further the feasibility of using this model in clinical practice by validating its performance in reducing the inter- and intra-observer variability of follicle diameter measurement. This study also investigated whether follicular area is a better biomarker than diameter in assessing follicular maturity. Data on 106 ovaries and 230 follicles collected from 80 cases of single follicular cycles and 26 cases of multiple follicular cycles constituted the validation set. Intra-observer variability was 0.973 and 0.982 for the senior sonographer and junior sonographer in single follicular cycles and 0.979 (0.971, 0.985) and 0.920 (0.892, 0.943) in multiple follicular cycles, respectively, while CR-Unet had no intra-group variation. Bland-Altman plot analysis indicated that the 95% limits of agreement between senior sonographer and CR-Unet (-2.1 to 1.1 mm, -2.02 to 0.75 mm) were smaller than those between senior sonographer and junior sonographer (-1.51 to 1.15 mm, -2.1 to 1.56 mm) in single and multiple follicular cycles. The average operating times of diameter measurement taken by the junior sonographer, senior sonographer and CR-Unet were 7.54 ± 1.8, 4.87 ± 0.84 and 1.66 ± 0.76 s, respectively (p < 0.001). Correlation analysis indicated that both manual and automated follicular area correlated better with follicular volume than diameter. The deep learning algorithm and the new biomarker of follicular area hold potential for clinical application of ultrasonic follicular monitoring.

Evolutionary sequences in mammalian reproductive biology.

Broad biological aspects and accepted evolutionary sequences may offer useful guidance towards a comprehensive explanation of the function of mammalian ovaries and oviducts and their vital contribution to the events of fertilization. Cooling of the preovulatory follicles before ovulation may well have its roots in the primitive stages of external fertilization in which aggregates of oocytes are shed into freshwater.

Tube-like structures within the ovarian follicle of Hydrolagus colliei indicate the early origins of follicle cell processes.

Follicle cell processes (FCP) are actin-based, tube-like structures that connect the developing oocyte to the follicle cells throughout oogenesis. They were first described in Selachians (sharks) where their suggested roles were facilitating the transport of metabolites to the developing oocyte and providing structural support to the large egg cells of sharks, an early stage in the evolution of viviparity. Subsequent studies found that FCP are absent in Rajiformes (skates), suggesting that FCP may have been novel structures specific to the sharks. Here, FCP in Hydrolagus colliei, a Chimaeriform, were described. The FCP of H. colliei differ morphologically from those previously described in sharks, but as they also contain actin, they presumably play similar roles provisioning the developing oocyte and providing structural support. The presence of FCP in the order Chimaeriformes suggests that their origin predates the split of the elasmobranchs and the holocephalans.

Increased number of large non-atretic follicles and co-dominance effects account for high litter sizes in Bonga sheep.

To understand the ovarian basis for prolificacy of Bonga sheep, a total of 31 ewes were selected based on litter size (LS) records and divided into two groups: High Prolificacy (HP) (n = 20) with LS ≥ 2 and Low Prolificacy (LP) (n = 11) with LS = 1. At a synchronized estrus, follicular dynamics were determined using transrectal ultrasonography. Plasma estradiol concentrations were also monitored. In total 27 ewes were observed in estrus being 9/11 LP (82%) and 18/20 HP (90%). On the day of estrus (day 0), the mean number of large follicles was higher (p < .05) in HP (1.78 ± 0.19) than in LP (1.0 ± 0.28) ewes. Prior to estrus, more (p < .05) medium follicles were visible for HP compared to LP ewes. Plasma estradiol concentrations were higher in HP compared to LP ewes (18.91 ± 0.41 vs. 14.51 ± 0.65 pg/ml; p < .05) and similarly was ovulation number (2.3 ± 0.15 vs. 1.28 ± 0. 14; p < .05). Higher ovulation rates and litter size in Bonga sheep are evidenced by the previous presence of more large follicles and the existence of co-dominance effects as most likely medium follicles are selected to ovulate.

The association between treatment parameters on the day of gonadotropin-releasing hormone antagonist initiation during a flexible protocol and oocyte maturation rate.

This study aimed to evaluate the effects of different treatment parameters on the day of GnRH antagonist initiation on oocyte maturation rate. We performed a retrospective cohort study of women aged ≤ 38 who underwent their first IVF-ICSI treatment using a flexible GnRH antagonist protocol in a single university-affiliated medical center during 2005-2015. Treatment parameters of three groups of oocyte maturation rates (<60%, 60-90%,>90%) were compared. Multivariate analysis was conducted to detect an association between treatment parameters on the day of GnRH antagonist initiation and oocyte maturation rate. The cohort included 458 patients, of whom 180 (39%) had a high oocyte maturation rate (≥90%), 211 (46%) had an oocyte maturation rate between 60-90% and 67 (15%) had a low maturation rate (≤60%). Women with a high maturation rate had longer duration of treatment (10.3 ± 2.9 days vs. 9.6 ± 2.5 vs. 9.5 ± 3.2, P = 0.019), lower levels of estradiol (1985 ± 1357 vs. 2406 ± 1666 vs. 2325 ± 1811, P = 0.027) and lower estradiol/maximal follicular diameter ratio on the day of GnRH antagonist initiation (137 ± 89 vs. 165 ± 103 vs. 163 ± 125, P = 0.019) as compared to women with medium and low maturation rates, respectively. Using linear regression multivariate analysis, lower estradiol and lower estradiol/maximal follicular diameter ratio on GnRH antagonist initiation day were associated with higher oocyte maturation rate. Further prospective studies to determine the best timing for GnRH antagonist initiation are needed.

Ovarian follicular response to oestrous synchronisation and induction of ovulation in Norwegian Red cattle.

BACKGROUND: Oestrous synchronisation of cattle has been widely applied to accomplish simultaneous ovulation in animals and facilitate timed artificial insemination. The main aim of this study was to investigate the ovarian follicular growth and ovulatory response to oestrus and ovulation synchronisation in Norwegian Red heifers and cows. Oestrous cycles in 34 heifers and 10 cows from 4 herds were synchronised with two PGF2α analogue treatments 11 days apart, followed by GnRH analogue treatment for induction of ovulation. Thereafter, the ovaries were examined by ultrasonography at 3 h intervals until ovulation. RESULTS: The luteolytic effect of the PGF2α analogue was verified in 9 of 10 cows by progesterone contents in milk. Maximum physical activity of the cows occurred on average 69 h after PGF2α analogue treatment. An ovulatory response was recorded in 95.5% (42/44) of the animals. A significant difference in follicle size at ovulation was found between 2 of the herds. Animals with medium sized and large follicles and heifers aged > 16 months ovulated earlier than other animals. CONCLUSIONS: The applied sequence of treatments in the study was shown to be effective in synchronizing and inducing ovulation within a relatively narrow time interval in the Norwegian Red heifers and cows, consistent with findings in other cattle breeds.

Ovary and ovarian bursa in dromedary camels: Clinical relevance of the topographical features.

This study aimed to associate ovarian characteristics with the efficiency of clinical examination and occurrence of genital diseases in dromedary camels. The reproductive tract of 870 female camels was examined through standard transrectal palpation and by ultrasonography during the breeding season. The ovaries were examined for structures and dimensions. The follicles were categorized according to size, the thickness of the wall and contents. Follicle aspiration was carried out from females with overgrown follicles (OVGF, n = 127), and the obtained follicular fluids were examined. At the slaughterhouse, 100 genital tracts were examined in situ and after dissection. Ovarian bursae were examined for patency and the presence of fluid (ovarian hydrobursitis, OVHB). Risks associated with the development of OVGF and OVHB were identified by the logistic regression. The results showed that, due to topographical difference, the right ovary was more accessible at rectal palpation than the left ovary (98.9% vs. 96.1%, p = .0005). Time needed for rectal palpation of the right ovary was shorter than the left ovary (25.1 ± 25 s vs. 34.6 ± 34.5 s, p = .03). Significant relationships were found between OVGF and OVHB (Odds ratio = 10.5, p = .001), OVGF and clinical endometritis (Odds ratio = 21.1, p = .001), OVGF and vaginal adhesion (Odds ratio = 4.4, p = .03), and OVHB and clinical endometritis (Odds ratio = 11.3, p = .001). Ultrasonographic examination was imperative for the differentiation between active corpus luteum, old non-active corpus luteum and small luteinized follicle. In conclusion, anatomical arrangement of the ovary and ovarian bursa in dromedary camels affects the likelihood of their accessibility during clinical examination and predisposes to unusual genital disorders.

Reproductive biology, morphology, and morphometry of ovaries and oviducts of Trachemys scripta elegans in Brazilian Cerrado / Biologia reprodutiva, morfologia e morfometriade ovários e ovidutos de Trachemys scripta elegans no Cerrado Brasileiro

Although Trachemys scripta elegans is an exotic species popular as a pet in Brazil, studies on reproductive biology and capacity are non-existent in the Brazilian Cerrado. This study analyzed ovarian and oviduct characteristics and the egg production capacity of T. scripta elegans grown in this biome. The findings will associate with the size of the specimens and the sexual maturity, aiming at comparisons with native and exotic populations, as well as interspecific and contributing to the understanding of its impact on the invaded ecosystems and the establishment of eradication programs. Thus, 39 females had evaluated the body biometry and the morphology and morphometry of the ovaries and oviducts. G2 (N=20): with Class I (>5-10mm) follicles, with Class I and Class II (>10-fold) follicles, 25mm) and G3 (N=9) with Class I, Class II and Class III (>25mm) follicles. Analysis of variance, Scott-Knott's test, and Pearson's correlation analysis showed that there was no significant difference between the groups in body biometry; in the mean gonadosomatic index and gonadal morphometry, only the width of the oviducts in the right antimer and the mass and width in the left antimer were higher in G3, the only one that presented eggs. There was positive and harmonic development between body mass, carapace, and plastron, and gonadal growth occurred concomitantly with body growth, indicating a higher reproductive potential and a positive relationship between the size of the litter and the female litter. The gonadosomatic index proved to be an excellent reproductive indicator, and the ovarian evaluation was a better indicator of sexual maturity than the maximum carapace length. Ovaries were irregular structures, without delimitation between the cortical and medullary regions and filled with vitelogenic follicles of different diameters, atresic follicles, and corpora lutea, which reflected the ovarian complexity of the species and the presence of follicular hierarchy. In the scarce stroma, two germinative beds were observed per ovary and the presence of gaps very close to the follicles and associated with the blood vessels. Analysis of gonadal tissue revealed three types of oocytes according to cytoplasmic characteristics: homogeneous, vesicular or vesicular in the cortex with apparent granules. Oviducts were functional and separated, joining only in the final portion to form the cloaca and subdivided into infundibulum, tuba, isthmus, uterus, and vagina. The structure of the uterine tube was composed of serosa, muscular and mucous, which was full of glands. The presence of eggs in the oviducts indicated that the specimens can reproduce in the Brazilian Cerrado. This study provides necessary and relevant information on the reproductive biology and capacity of T. scripta elegans in the Brazilian Cerrado and can contribute to the understanding of its impact on the invaded ecosystems and the establishment of eradication programs. The extraction of females with capacity can reduce the annual reproductive yield of the species and decrease its effect on local biodiversity.(AU)

Embora Trachemys scripta elegans seja uma espécie exótica popular como animal de estimação no Brasil, estudos sobre biologia e capacidade reprodutivas são inexistentes no Cerrado brasileiro. Este estudo analisou características ovarianas e do oviduto e a capacidade de produção de ovos em T. scripta elegans criadas neste bioma, correlacionando estes achados ao tamanho dos espécimes e a maturidade sexual, visando comparações com populações nativas e exóticas, bem como interespecíficas e contribuir para a compreensão de seu impacto nos ecossistemas invadidos e com o estabelecimento de programas de erradicação. Assim, 39 fêmeas tiveram avaliadas a biometria corporal e a morfologia e morfometria dos ovários e ovidutos. De acordo com o tamanho dos folículos ovarianos as fêmeas foram separadas em G1 (N= 10): com folículos Classe I (>5-10 mm), G2 (N= 20): com folículos Classe I e Classe II (>10-25 mm) e G3 (N= 9) com folículos Classe I, Classe II e Classe III (>25 mm). À análise de variância, teste de Scott-Knott e à análise de correlação de Pearson verificou-se que não houve diferença significativa entre os grupos na biometria corporal; no índice gonadossomático médio e na morfometria gonadal, apenas a largura dos ovidutos no antímero direito e a massa e a largura no antímero esquerdo foram maiores no G3, o único que apresentou ovos. Houve desenvolvimento positivo e harmônico entre massa corporal, carapaça e plastrão e o crescimento gonadal ocorreu concomitante ao crescimento corporal, indicando maior potencial reprodutivo e relação positiva entre o tamanho da ninhada de ovos e o da fêmea. O índice gonadossomático mostrou-se um bom indicador reprodutivo e a avaliação ovariana um melhor indicador da maturidade sexual que o comprimento máximo da carapaça. Ovários foram estruturas irregulares, sem delimitação entre a região cortical e medular e repletos de folículos vitelogênicos de diferentes diâmetros, folículos atrésicos e corpos lúteos, que refletiram a complexidade ovariana da espécie e a presença de hierarquia folicular. No estroma escasso foram observados dois leitos germinativos por ovário e a presença de lacunas muito próximas aos folículos e associadas aos vasos sanguíneos. A análise do tecido gonadal revelou três tipos de oócitos de acordo com as características do citoplasma: homogêneo, vesicular ou vesicular no córtex com grânulos aparentes. Ovidutos eram funcionais e separados, unindo-se apenas na porção final para formar a cloaca e subdividiam-se em infundíbulo, tuba uterina, istmo, útero e vagina. A estrutura da tuba uterina era constituída de serosa, muscular e mucosa, a qual era repleta de glândulas. A presença de ovos nos ovidutos indicou que os espécimes podem se reproduzir no cerrado brasileiro. Este estudo fornece informações básicas e relevantes da biologia e capacidade reprodutivas de T. scripta elegans no Cerrado brasileiro e pode contribuir com a compreensão de seu impacto nos ecossistemas invadidos e com o estabelecimento de programas de erradicação, uma vez que a extração de fêmeas com capacidade reprodutiva pode contribuir com a diminuição do rendimento reprodutivo anual da espécie e diminuir seu efeito sobre a biodiversidade local.(AU)

Slow Freezing Versus Vitrification of Mouse Ovaries: from Ex Vivo Analyses to Successful Pregnancies after Auto-Transplantation.

Slow freezing (SF) is the reference method for ovarian tissue cryopreservation. Vitrification (VT) constitutes an alternative but controversial method. This study compares SF and VT (open [VTo] and closed [VTc] systems) in terms of freezing damage and fertility restoration ability. In vitro analyses of C57Bl/6 SF or VTo-ovaries, immediately after thawing/warming or after culture (cult), revealed that event though follicular density was similar between all groups, nuclear density was decreased in VTo-ovaries compared to CT-ovaries (CT = 0.50 ± 0.012, SF = 0.41 ± 0.03 and VTo = 0.29 ± 0.044, p < 0.01). Apoptosis was higher in VTo-cult ovaries compared to SF-cult ovaries (p < 0.001) whereas follicular Bmp15 and Amh gene expression levels were decreased in the ovaries after culture, mostly after VTo (p < 0.001). Natural mating after auto-transplantation of SF, VTo and VTc-ovaries revealed that most mice recovered their oestrous cycle. Fertility was only restored with SF and VTo ovaries (SF: 68%; VTo: 63%; VTc: 0%; p < 0.001). Mice auto-transplanted with SF and VTo-ovaries achieved the highest number of pregnancies. In conclusion, in vitro, no differences between SF and VTo were evident immediately after thawing/warming but VTo ovaries displayed alterations in apoptosis and follicular specific proteins after culture. In vivo, SF and VTo ovary auto-transplantation fully restored fertility whereas with VTc-ovary auto-transplantation no pregnancies were achieved.

Effect of somatotropin on survival and diameter of bovine preantral follicles / Efeito da somatotropina na sobrevivência e no diâmetro de folículos pré-antrais de bovinos

The aim of this study was to evaluate the effect of Recombinant bovine somatotropin (rbST) on survival and diameter of bovine preantral ovarian follicles (PAOF) cultured in vitro. Ovaries were collected from adult cows and fragments of ovarian cortex were immediately fixed (non-cultured control) or cultured in vitro in α-MEM+ alone or containing 10, 50, 100 or 1,000ng/mL rbST. The fragments were processed for Classical Histology and Transmission Electron Microscopy. After one and seven days of culture, the percentage of normal follicles in the non-cultured control was superior (P< 0.05) to the follicles cultured in α-MEM+ alone or with different rbST concentrations. The oocyte and follicular mean diameter did not increase during the culture for one and seven days, both in media containing rbST and in the medium without this hormone. The only medium in which there was no reduction in follicular diameter with the time of culture was the medium without rbST. Ultrastructural damage in PAOF cultured in vitro was found. It is concluded that the use of rbST at different concentrations in in situ culture of bovine preantral follicles has no beneficial effects on survival and growth of bovine PAOF.(AU)

O objetivo deste trabalho foi avaliar o efeito da somatotropina recombinante bovina (rbST) sobre a sobrevivência e o diâmetro de folículos ovarianos pré-antrais (FOPA) bovinos cultivados in vitro. Ovários foram coletados de vacas adultas e fragmentos do córtex ovariano foram imediatamente fixados (controle não cultivado) ou cultivados in vitro em α-MEM + sozinho ou contendo 10, 50, 100 ou 1.000ng/mL de rbST. Os fragmentos foram processados para histologia clássica e microscopia eletrônica de transmissão. Após um e sete dias de cultivo, o percentual de folículos normais no controle não cultivado foi superior (P<0,05) aos cultivados em α-MEM + sozinho ou acrescido de diferentes concentrações de rbST. Os diâmetros médios oocitário e folicular não aumentaram durante o cultivo por um e sete dias, tanto nos meios contendo rbST, como no meio sem esse hormônio (α-MEM + ). O único meio em que não houve redução no diâmetro folicular com o tempo de cultivo foi o sem rbST. Verificaram-se ainda danos ultraestruturais em FOPA cultivados in vitro. Conclui-se que o uso de rbST em diferentes concentrações no cultivo in situ de folículos pré-antrais bovinos não tem efeitos benéficos na sobrevivência e no crescimento de FOPA bovinos.(AU)

Brangus cows have ovarian reserve parameters more like Brahman than Angus cows.

Bos indicus females have more surface antral follicles than Bos taurus females; however, histological studies demonstrated no difference in total number of primordial follicles between these two biological types of cattle. Primordial follicle density in the ovary was less in Nelore ovaries compared to Angus ovaries, but no studies have examined the primordial follicle density in Bos indicus cross-bred females. It, therefore, was hypothesized that primordial follicle density in the ovary would decrease as percentage Bos indicus increased. Ovaries were collected from cross-bred Angus (n = 32, no Bos indicus influence), Brangus (n = 15), or Brahman (n = 9) cows and prepared for histological evaluation. There was no difference in total number of primordial follicles per ovary between breeds (P > 0.10). When numbers of primordial follicles were expressed on a per gram of ovarian tissue basis, there were fewer primordial follicles per gram of ovarian tissue in Brangus and Brahman cows than in Angus cows (P < 0.05). Brangus cows did not differ from Brahman cows in primordial follicle density (P > 0.10). Differences in primordial follicle density could indicate differences in capacity of ovarian stroma to produce factors necessary for oogonial proliferation and primordial follicle formation among breeds. Identifying these factors could improve the aprroach for culturing pre-antral follicles of cattle. Furthermore, these results explain why ultrasonographic antral follicle counts may need to be adjusted to a greater threshold to predict size of the ovarian reserve and determine ovarian reserve related reproductive traits in Bos indicus females.

Morphological patterns of cell death in ovarian follicles of primary and secondary growth and postovulatory follicle complex of the annual killifish Millerichthys robustus (Cyprinodontiformes: Cynolebiidae).

The dynamics of cellular development and homeostasis of the ovary depend on the balance between proliferation and cell death throughout the reproductive cycle. Millerichthys robustus is an annual fish whose ovarian follicles develop asynchronously, allowing daily reproduction from sexual maturity until death. The objective of this research is to describe, histologically, the processes of follicular atresia and regression of postovulatory follicular complexes (POC) throughout a reproductive cycle of M. robustus. Patterns of cell death were documented by apoptosis in atretic follicles and POC, and necrosis in the POC after ovulation with an associated inflammatory response. Atretic follicles were seen from the onset of sexual maturity, during week three post-hatching (PH), both in primary growth (from the Cortical alveoli step, with folliculogenesis completed) and secondary growth Stages, with a higher prevalence in the latter. POCs were observed in different stages of regression from week four PH until the death of the fish. The apoptotic characteristics found were: (i) fragmentation of the nuclear membrane and zona pellucida, and liquefaction of the cortical alveoli and yolk; (ii) follicular cells becoming phagocytic, increasing their size, and migrating within the oocyte; and (iii) formation of an intrafollicular lumen, a product of phagocytosis of the oocyte constituents and dispersed pigments that remain after the digestion of yolk and cortical alveoli. The morphological changes of the follicular cells of the POC, from a squamous morphology after ovulation to columnar during its regression with PAS+ contents, was documented, suggesting a secretory activity.

First characterization of preantral follicles in the ovaries of adult alpaca / Primera caracterización de folículos preantrales de ovario de alpaca adulta

It is a fact that while even basic reproductive information on alpacas is unavailable, the normal ovarian reserve of this species in comparison to other species is also unidentified. In this study, the ovarian preantral follicles in healthy adult alpacas were characterized in order to establish a general model to in vitro studies. Ten ovaries were collected from five adult alpacas. The ovarian cortex samples were fixed with paraformaldehyde and histological analysis was done. Normal and degenerated follicles percentages were determined. The normal follicles were measured and classified in primordial, transitional, primary and secondary stages. Most of the preantral follicles present in the ovarian cortex of alpacas were primordial and transitional stages; primary (6.10%) and secondary (0.37%) follicles were rarely found. The primary and secondary follicles were larger in diameter when compared with the primordial and transitional follicles. The largest oocyte diameter was recorded in the secondary follicles (P < 0.05). This study serves to establish a biological model for future reproduction studies in Alpacas or as possible biological model for studies of folliculogenesis in humans(AU)

Es un hecho que, si bien no se dispone de información reproductiva básica sobre las alpacas, la reserva ovárica normal de esta especie en comparación con otras especies tampoco está identificada. En este estudio, se caracterizaron los folículos preantrales ováricos en alpacas adultas sanas. Se recogieron diez ovarios de cinco alpacas adultas. Las muestras de la corteza ovárica se fijaron con paraformaldehído y se realizó un análisis histológico. Se determinaron los porcentajes de folículos normales y degenerados. Los folículos normales se midieron y clasificaron en estadios: primordiales, de transición, primarios y secundarios. La mayoría de los folículos preantrales presentes en la corteza ovárica de las alpacas eran estadios primordiales y de transición; Raras veces se encontraron folículos primarios (6.10%) y secundarios (0.37%). Los folículos primarios y secundarios tenían un diámetro mayor en comparación con los folículos primordiales y de transición. El mayor diámetro de ovocitos se registró en los folículos secundarios (P <0.05). Este estudio sirve para establecer un modelo biológico para futuros estudios de reproducción en alpacas o como posible modelo biológico para estudios de foliculogénesis en humanos(AU)