Effect of carvacrol antioxidant capacity on oocyte maturation and embryo production in cattle.

Carvacrol (C10H14O), an efficient phenolic antioxidant substance for several cell types, may become a useful antioxidant for female germ cells and embryo culture. This study investigates the effects of carvacrol supplementation on bovine oocytes in in vitro maturation (IVM) and embryo production. In total, 1222 cumulus-oocyte complexes were cultured in TCM-199+ alone (control treatment) or supplemented with carvacrol at the concentrations of 3 µM (Carv-3), 12.5 µM (Carv-12.5), or 25 µM (Carv-25). After IVM, the oocytes were subjected to in vitro fertilization and embryo production, and the spent medium post-IVM was used for evaluating the levels of reactive oxygen species and the antioxidant capacity (2,2-diphenyl-1-picryl-hydrazyl-hydrate and 2,2'-azinobis-3-ethyl-benzothiozoline-6-sulphonic acid quantification). A greater (P < 0.05) antioxidant potential was observed in the spent medium of all carvacrol-treated groups compared with the control medium. Moreover, the addition of carvacrol to the maturation medium did not affect (P > 0.05) blastocyst production on days 7 and 10 of culture; however, the total number of cells per blastocyst was reduced (P < 0.05) in two carvacrol-treated groups (Carv-3 and Carv-25). In conclusion, carvacrol demonstrated a high antioxidant capacity in the spent medium after oocyte maturation; however, although embryo production was not affected, in general, carvacrol addition to IVM medium reduced the total number of cells per blastocyst. Therefore, due to the high antioxidant capacity of carvacrol, new experiments are warranted to investigate the beneficial effects of lower concentrations of carvacrol on embryo production in cattle and other species.

Seasonal variation in equine follicular fluid proteome.

BACKGROUND: Proteomic studies of follicular fluid (FF) exist for several species, including the horse; however, the seasonal influence on FF proteome has not been explored in livestock. The application of high-throughput proteomics of FF in horse has the potential to identify seasonal variations of proteins involved in follicle and oocyte growth. METHODS: This study (i) profiles the proteomes of equine FF collected from dominant growing follicles during the spring anovulatory season (SAN), and spring (SOV), summer (SUM), and fall (FOV) ovulatory seasons; and (ii) identifies season-dependent regulatory networks and associated key proteins. RESULTS: Regardless of season, a total of 90 proteins were identified in FF, corresponding to 63, 72, 69, and 78 proteins detected in the SAN, SOV, SUM, and FOV seasons, respectively. Fifty-two proteins were common to all seasons, a total of 13 were unique to either season, and 25 were shared between two seasons or more. Protein-to-protein interaction (PPI) analysis indicated the likely critical roles of plasminogen in the SAN season, the prothrombin/plasminogen combination in SUM, and plasminogen/complement C3 in both SOV and FOV seasons. The apolipoprotein A1 appeared crucial in all seasons. The present findings show that FF proteome of SUM differs from other seasons, with FF having high fluidity (low viscosity). CONCLUSIONS: The balance between the FF contents in prothrombin, plasminogen, and coagulation factor XII proteins favoring FF fluidity may be crucial at the peak of the ovulatory season (SUM) and may explain the reported lower incidence of hemorrhagic anovulatory follicles during the SUM season.

Follicle growth and endocrine dynamics in women with spontaneous luteinized unruptured follicles versus ovulation.

STUDY QUESTION: Do growth patterns and endocrine profiles differ between ovulatory follicles (OvFs) and luteinized unruptured follicles (LUFs) in women? SUMMARY ANSWER: Growth rates, diameters and associated endocrine profiles differed between OvFs and LUFs in unstimulated cycles. WHAT IS KNOWN ALREADY: Two-three waves of antral follicles develop during the menstrual cycle in ovulatory women of reproductive age, with the second or third wave terminating in ovulation. In contrast, some women can develop LUFs, where a preovulatory follicle fails to rupture and there is subsequent luteinization of the follicle wall. However, no study has compared OvFs and LUFs in unstimulated cycles. STUDY DESIGN, SIZE, DURATION: This retrospective observational study was conducted in 56 healthy women of reproductive age (range: 19-41 years) and with a history of regular menstrual cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants who met inclusion criteria were enrolled, as previously reported. Daily transvaginal ultrasonography was performed for one interovulatory interval (IOI) to measure the diameters of all follicles >2 mm. Blood samples were collected every 3 days during the IOI to measure serum concentrations of FSH, LH, estradiol and progesterone. MAIN RESULTS AND THE ROLE OF CHANCE: The interval from emergence to deviation (i.e. follicle selection) was shorter (P < 0.05) for LUFs compared to OvFs. However, the intervals from emergence to maximum diameter and deviation to maximum diameter were longer (P < 0.05) for LUFs compared to OvFs. Follicle deviation in LUFs occurred at a larger diameter (P < 0.05) compared to OvFs, and LUFs grew to larger (P < 0.0001) diameters compared to OvFs. Moreover, LUFs grew faster (P < 0.05) from emergence to deviation and from deviation to maximum diameter, compared to OvFs. LUFs were associated with low (P < 0.05) systemic LH levels at emergence and maximum diameter compared to OvFs. LUFs were also associated with low (P < 0.05) systemic FSH and high (P < 0.05) systemic progesterone at deviation and maximum diameter, respectively. Estradiol was higher (P < 0.05) at deviation and lower (P < 0.05) at maximum diameter for LUFs compared to OvFs. LIMITATIONS, REASONS FOR CAUTION: A 3-day interval of blood sampling for hormonal analyses was conducted, as a more frequent sampling interval was not considered acceptable by the study volunteers. A 3-day sampling interval did not allow characterization of acute changes in hormone production during the IOI. In addition, study visits were less frequent when LUFs persisted long after the expected day of the second ovulation of the IOI. WIDER IMPLICATIONS OF THE FINDINGS: Information about the growth and endocrine dynamics of OvFs and LUFs developing in unstimulated cycles in women may be applied to the early detection of LUF-associated anovulatory infertility and clinical management of women with this condition. STUDY FUNDING/COMPETING INTEREST(S): No external funding sources were used for this study. The authors have no conflicts of interest in publishing this manuscript. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Identifier: NCT01389141.

In vivo antral follicle wall biopsy: a new research technique to study ovarian function at the cellular and molecular levels.

BACKGROUND: In vivo studies involving molecular markers of the follicle wall associated with follicular fluid (FF) milieu are crucial for a better understanding of follicle dynamics. The inability to obtain in vivo samples of antral follicle wall (granulosa and theca cells) without jeopardizing ovarian function has restricted advancement in knowledge of folliculogenesis in several species. The purpose of this study in mares was to develop and validate a novel, minimally invasive in vivo technique for simultaneous collection of follicle wall biopsy (FWB) and FF samples, and repeated collection from the same individual, during different stages of antral follicle development. We hypothesized that the in vivo FWB technique provides samples that maintain the normal histological tissue structure of the follicle wall layers, offers sufficient material for various cellular and molecular techniques, and allows simultaneous retrieval of FF. METHODS: In Experiment 1 (ex vivo), each follicle was sampled using two techniques: biopsy forceps and scalpel blade (control). In Experiment 2 (in vivo), FWB and FF samples from 10-, 20-, and 30-mm follicles were repeatedly and simultaneously obtained through transvaginal ultrasound-guided technique. RESULTS: In Experiment 1, the thickness of granulosa, theca interna, and theca externa layers was not influenced (P > 0.05) by the harvesting techniques. In Experiment 2, the overall recovery rates of FWB and FF samples were 97 and 100%, respectively. However, the success rate of obtaining samples with all layers of the follicle wall and clear FF varied according to follicle size. The expression of luteinizing hormone receptor (LHR) was mostly confined in the theca interna layer, with the estradiol-related receptor alpha (ERRα) in the granulosa and theca interna layers. The 30-mm follicle group had greater (P < 0.05) LHR expression in the theca interna and ERRα in the granulosa layer compared to the other groups. The overall expression of LHR and ERRα, and the intrafollicular estradiol were higher (P < 0.05 - P < 0.0001) in the 30-mm follicle group. CONCLUSION: The in vivo technique developed in this study can be repeatedly and simultaneously used to provide sufficient FWB and FF samples for various cellular and molecular studies without jeopardizing the ovarian function, and has the potential to be translated to other species, including humans.

Ovarian fragment sizes affect viability and morphology of preantral follicles during storage at 4°C.

The method of transportation and the conditions imposed on the ovarian tissue are pivotal aspects for the success of ovarian tissue cryopreservation (OTC). The aim of this study was to evaluate the effect of the size of the ovarian tissue (e.g. whole ovary, biopsy size and transplant size) during different times of storage (0, 6, 12 and 24 h) on the structural integrity of equine ovarian tissue transported at 4°C. Eighteen pairs of ovaries from young mares (<10 years old) were harvested in a slaughterhouse and processed to simulate the fragment sizes (biopsy and transplant size groups) or kept intact (whole ovary group) and stored at 4°C for up to 24 h in α-MEM-enriched solution. The effect of the size of the ovarian tissue was observed on the morphology of preantral follicles, stromal cell density, DNA fragmentation and mitochondrial membrane potential. The results showed that (i) biopsy size fragments had more morphologically normal preantral follicles after 24 h of storage at 4°C; (ii) mitochondrial membrane potential was the lowest during each storage time when the whole ovary was used; (iii) DNA fragmentation rate in the ovarian cells of all sizes of fragments increased as storage was prolonged and (iv) transplant size fragments had increased stromal cell density during storage at cool temperature. In conclusion, the biopsy size fragment was the best to preserve follicle morphology for long storage (24 h); however, transportation/storage should be prior determined according to the distance (time of transportation) between patient and reproduction centers/clinics.

Preantral follicle density in ovarian biopsy fragments and effects of mare age.

The aims of the present study were to: (1) evaluate preantral follicle density in ovarian biopsy fragments within and among mares; (2) assess the effects of mare age on the density and quality of preantral follicles; and (3) determine the minimum number of ovarian fragments and histological sections needed to estimate equine follicle density using a mathematical model. The ovarian biopsy pick-up method was used in three groups of mares separated according to age (5-6, 7-10 and 11-16 years). Overall, 336 preantral follicles were recorded with a mean follicle density of 3.7 follicles per cm2. Follicle density differed (P<0.05) among animals, ovarian fragments from the same animal, histological sections and age groups. More (P<0.05) normal follicles were observed in the 5-6 years (97%) than the 11-16 years (84%) age group. Monte Carlo simulations showed a higher probability (90%; P<0.05) of detecting follicle density using two experimental designs with 65 histological sections and three to four ovarian fragments. In summary, equine follicle density differed among animals and within ovarian fragments from the same animal, and follicle density and morphology were negatively affected by aging. Moreover, three to four ovarian fragments with 65 histological sections were required to accurately estimate follicle density in equine ovarian biopsy fragments.

Follicle vascularity coordinates corpus luteum blood flow and progesterone production.

Colour Doppler ultrasonography was used to compare the ability of preovulatory follicle (POF) blood flow and its dimensions to predict the size, blood flow and progesterone production capability of the subsequent corpus luteum (CL). Cows (n=30) were submitted to a synchronisation protocol. Follicles ≥7mm were measured and follicular wall blood flow evaluated every 12h for approximately 3.5 days until ovulation. After ovulation, cows were scanned daily for 8 days and similar parameters were evaluated for the CL. Blood samples were collected and plasma progesterone concentrations quantified. All parameters were positively correlated. Correlation values ranged from 0.26 to 0.74 on data normalised to ovulation and from 0.31 to 0.74 on data normalised to maximum values. Correlations between calculated ratios of both POF and CL in data normalised to ovulation and to maximum values ranged from moderate (0.57) to strong (0.87). Significant (P<0.0001) linear regression analyses were seen in all comparisons. In conclusion, higher correlations were observed between the dimensions of POF and/or CL and blood flow of both structures, as well as POF and/or CL blood flow with plasma progesterone concentrations of the resultant CL. These findings indicate that follicle vascularity coordinates CL blood flow and progesterone production in synchronised beef cows.

The mare as a model for luteinized unruptured follicle syndrome: intrafollicular endocrine milieu.

Luteinized unruptured follicle (LUF) syndrome is a recurrent anovulatory dysfunction that affects up to 23% of women with normal menstrual cycles and up to 73% with endometriosis. Mechanisms underlying the development of LUF syndrome in mares were studied to provide a potential model for human anovulation. The effect of extended increase in circulating LH achieved by administration of recombinant equine LH (reLH) or a short surge of LH and decrease in progesterone induced by prostaglandin F2α (PGF2α) on LUF formation (Experiment 1), identification of an optimal dose of COX-2 inhibitor (flunixin meglumine, FM; to block the effect of prostaglandins) for inducing LUFs (Experiment 2), and evaluation of intrafollicular endocrine milieu in LUFs (Experiment 3) were investigated. In Experiment 1, mares were treated with reLH from Day 7 to Day 15 (Day 0=ovulation), PGF2α on Day 7, or in combination. In Experiment 2, FM at doses of 2.0 or 3.0 mg/kg every 12 h and human chorionic gonadotropin (hCG) (1500 IU) were administered after a follicle ≥32 mm was detected. In Experiment 3, FM at a dose of 2.0 mg/kg every 12 h plus hCG was used to induce LUFs and investigate the intrafollicular endocrine milieu. No LUFs were induced by reLH or PGF2α treatment; however, LUFs were induced in 100% of mares using FM. Intrafollicular PGF2α metabolite, PGF2α, and PGE2 were lower and the ratio of PGE2:PGF2α was higher in the induced LUF group. Higher levels of intrafollicular E2 and total primary sex steroids were observed in the induced LUF group along with a tendency for higher levels of GH, cortisol, and T; however, LH, PRL, VEGF-A, and NO did not differ between groups. In conclusion, this study reveals part of the intrafollicular endocrine milieu and the association of prostaglandins in LUF formation, and indicates that the mare might be an appropriate model for studying the poorly understood LUF syndrome.

Long-term in vitro culture of ovarian cortical tissue in goats: effects of FSH and IGF-I on preantral follicular development and FSH and IGF-I receptor mRNA expression.

Long-term in vitro culture (16 days) of caprine ovarian cortical tissue was performed to test the effect of FSH and IGF-I on the viability and development of preantral follicles and mRNA expression for FSH and IGF-I receptors. Fragments were cultured in α-MEM(+) alone or supplemented with different combinations of FSH and IGF-I (sequential medium). The culture period was divided into two parts. Follicles were isolated and classified as normal or abnormal and primordial, primary or secondary. Viability of isolated follicles was determined by staining with Trypan Blue dye. Expression of FSHR and IGFR-1 mRNA was evaluated by qPCR. At day 8 of culture, more (P < 0.05) follicles in treatments containing IGF-I alone or associated with FSH were normal and viable (overall mean, 81 % and 79 % respectively) than the treatments cultured with FSH or α-MEM(+) alone (68 % and 63 %). At day 16 of culture, treatments with FSH and/or IGF-I had more (P < 0.05) viable follicles (69 %) than α-MEM(+) (38 %). The percentages of follicular development observed in the IGF-I/FSH, FSH+IGF-I/FSH+IGF-I and FSH/IGF-I treatments were similar but higher (P < 0.05) than the other treatments. FSH and IGF-I during the entire culture period maximized (P < 0.05) follicular and oocyte diameters and the percentage of secondary follicles (28 %). FSHR mRNA expression in the non-cultured control was similar to the treatment supplemented with FSH and IGF-I but higher (P < 0.05) than α-MEM(+). IGFR-1 expression did not differ among treatments. Association of FSH and IGF-I in long-term in vitro culture promoted follicular development, maintaining FSHR mRNA expression.

In vitro- and in vivo-derived early antral follicles have comparable in vitro follicular growth and oocyte maturation rates in goats.

Recent in vitro follicle culture (IVFC) studies in caprine have yielded lower maturation rates using late preantral follicles compared to early antral follicles. Thus, research focusing on developing stage-specific customized culture systems able to improve the efficiency of IVFC for late preantral follicles are warranted. This study aimed to compare the morphometric features, estradiol production, and gene expression between early antral caprine follicles produced in vitro and in vivo. In vitro-derived early antral follicles were produced after a 6-day in vitro culture of late preantral follicles, while in vivo-derived early antral follicles were yielded immediately after isolation from the ovaries; antral follicles were, thereafter, cultured for 18 days. In vitro-derived antral follicles were cultured either in a medium developed for preantral follicles (PF medium) or in a medium developed for antral follicles (AF medium). In vivo-derived early antral follicles, on the other hand, were cultured in AF medium (Control treatment). Results demonstrated that in vitro-derived antral follicles cultured in PF medium produced higher estradiol concentration, and m-RNA expression for matrix metalloproteinase-9 (MMP-9), and insulin receptor when compared to both in vitro- and in vivo-derived antral follicles cultured in AF medium. Remarkably, in vitro-derived antral follicles cultured in PF medium had similar MII and oocytes ≥110 µm rates compared with in vivo-derived antral follicles (Control treatment). In conclusion, when cultured in a single and appropriate medium (i.e., PF medium), in vitro-derived early antral follicles had comparable oocyte maturation rates to the in vivo-derived early antral follicles.

Oocyte in vitro maturation with eugenol improves the medium antioxidant capacity and total cell number per blastocyst.

This study investigates the impact of eugenol (EU) supplementation on bovine oocyte in vitro maturation (IVM) and antioxidant capacity, as well as in vitro embryo production and quality after conventional in vitro fertilization (IVF). A total of 1077 cumulus oocyte complexes were cultured in TCM-199+ without EU supplementation (control treatment) or supplemented with EU at the concentrations of 10 µM (EU-10), 20 µM (EU-20), or 40 µM (EU-40). After IVM, the oocytes were subjected to IVF and embryo culture. The addition of EU at 40 µM to the IVM medium improved (P < 0.05) the antioxidant capacity and cleavage rate when compared to the control treatment. Moreover, a positive correlation (r = 0.61, P < 0.03) was observed between cleavage rate and EU concentration. The addition of EU at concentrations of 10 and 20 µM decreased (P < 0.05) the calreticulin (CALR) levels in expanded blastocysts when compared to the control treatment and EU-40 treatment. However, the EU-10 and EU-20 treatments had a greater (P < 0.05) mean total cell number (TCN) per expanded blastocyst when compared to the control treatment and EU-40 treatment. In conclusion, the addition of EU to the enriched culture medium during IVM of bovine oocytes improved the antioxidant capacity of the spent medium, as well as the cleavage rate and embryonic quality (i.e., TCN/expanded blastocyst), and reduced the endoplasmic reticulum stress (i.e., CALR levels) in the embryos. Thus, we recommend enriching the IVM medium with 10 µM EU for in vitro bovine embryo production.

Impact of ethanol and heat stress-dependent effect of ultra-diluted Arnica montana 6 cH on in vitro embryo production in cattle.

This study evaluated the effect of adding ultra-diluted and dynamized Arnica montana 6 cH, and its vehicle (0.3% ethanol) to the in vitro maturation (IVM) medium, in the absence (experiment 1) or presence (experiment 2) of heat stress (HS), on bovine oocyte maturation and in vitro embryo production (IVEP). In experiment 1 (n = 902 cumulus oocyte complexes, COCs), the treatments were 1) IVM medium (Control treatment), 2) IVM medium + 0.3% ethanol, and 3) IVM medium + Arnica montana 6 cH. In experiment 2 (n = 1064 COCs), the treatments were 1) IVM medium without HS, 2) IVM medium under HS, 3) IVM medium + ethanol under HS, and 4) IVM medium + Arnica montana under HS. In the absence of HS (experiment 1), the addition of Arnica montana to the IVM medium had a deleterious effect on the IVEP (cleavage and blastocyst rates) and the total cell number/blastocysts. On the other hand, ethanol (0.3%) increased IVEP in relation to the Control and Arnica montana treatments. However, in the presence of HS during IVM (experiment 2), the addition of ethanol or Arnica montana increased IVEP when compared to the HS treatment alone, and the Arnica montana treatment resulted in greater total cell number/blastocysts compared to the other treatments. In conclusion, this study showed for the first time that the negative or positive effect of Arnica montana 6 cH on IVEP depends on the culture condition (i.e., absence or presence of HS during IVM). On the other hand, ethanol showed beneficial and consistent results on IVEP regardless of exposure to HS.

Heterotopic autotransplantation of equine ovarian tissue using intramuscular versus subvulvar grafting sites: Preliminary results.

Ovarian tissue transplantation (OTT) is a technique well established and successfully applied in humans using mainly orthotopic or heterotopic transplantation sites. In livestock, OTT is still in its infancy and, therefore, different aspects of the technique, including the efficiency of different heterotopic OTT sites as well as the potential effect of age (i.e., young vs. old mares) in the ovarian graft quality, need to be investigated. The present study investigated the efficacy of the intramuscular (IM) or the novel subvulvar mucosa (SV) heterotopic autotransplantation sites to maintain the survivability of the grafts for 3 and 7 days post-OTT. Ovarian biopsy fragments were obtained in vivo and distributed to the following treatments: Fresh control group (ovarian fragments immediately fixed), SV-3, IM-3, SV-7, and IM-7. During and after graft harvesting, the macroscopic characteristics of the grafts (i.e., adherence, morphology, and bleeding) were scored, and the percentages of morphologically normal and developing preantral follicles as well as the follicular and stromal cell densities of the grafts were evaluated. The results were that similar (P > 0.05) macroscopic scores were observed between both transplantation sites 7 days post-OTT, with positive correlations (P < 0.01) found among adherence, morphology, and bleeding of the grafts. A lower (P < 0.05) percentage of morphologically normal follicles was found 7 days post-OTT in the SV site (82%) compared with the Fresh control group (99%) and IM site (95%); however, the percentages of developing follicles were similar (P > 0.05) between both transplantation sites 7 days post-OTT (30-43%). Although similar (P > 0.05) follicular densities were found in both transplantation sites in young and old mares at 3 and 7 days post-OTT, large individual variation in the follicular depletion rate was observed regardless of transplantation site. The Fresh control group and SV-7 treatments had higher (P < 0.05) stromal cell densities in young and old mares compared with both IM-7 treatments. When comparing transplant sites between young and old mares, the follicular density in old mares and the stromal cell density in young mares were greater (P < 0.05) in the SV than in the IM site. In conclusion, even though the transplantation sites differentially affected some end points, overall comparable findings of the OTT technique using both heterotopic autotransplantation sites (i.e., IM and SV) for equine ovarian tissue were observed.

Temporal relationships of the LH surge and ovulation to echotexture and power Doppler signals of blood flow in the wall of the preovulatory follicle in heifers.

Changes in echotexture and blood flow in the wall of preovulatory follicles in heifers were studied in relation to the LH surge and ovulation in gonadotrophin-releasing hormone-induced (n = 7; Experiment 1) and spontaneous (n = 8; Experiment 2) ovulators. Ultrasonographic examinations and blood sampling were performed either every hour (Experiment 1) or every 6 h (Experiment 2). The interval from LH peak to ovulation in induced and spontaneous ovulators was 27.1 +/- 0.3 and 34.5 +/- 1.5 h, respectively. Follicle diameter did not increase between the LH peak and ovulation. In the induced ovulators, serration of the stratum granulosum was detected in one (14%), two (29%), three (43%) and four (57%) heifers at 4, 3, 2 and 1 h before ovulation, respectively. An initial increase in blood flow (P < 0.001) encompassed the LH peak in both experiments. In the induced ovulators, blood flow increased (P < 0.02) to maximum 3 h after the LH peak, maintained a plateau for 5 h, decreased (P < 0.05) between 9 and 14 h, increased (P < 0.05) again between 19 and 21 h and then decreased (P < 0.01) between 25 and 26 h (1 h before ovulation). The biphasic increase and decrease in blood flow and serration of the granulosum in the wall of the preovulatory follicle in cattle are novel findings.

Harvesting, processing, and evaluation of in vitro-manipulated equine preantral follicles: A review.

The mammalian ovary is responsible for essential stages of folliculogenesis and hormonal production, regulating the female physiological functions during the menstrual/estrous cycles. The mare has been considered an attractive model for comparative studies due to the striking similarities shared with women regarding in vivo and in vitro folliculogenesis. The ovarian follicular population in horses contains a large number of oocytes enclosed in preantral follicles that are yet to be explored. Therefore, the in vitro manipulation of equine preantral follicles aims to avoid the process of atresia and promote the development of follicles with competent oocytes. In this regard, after ovarian tissue harvesting, the use of appropriate processing techniques, as well as suitable approaches to evaluating equine preantral follicles and ovarian tissue, are necessary. Although high-quality equine ovarian tissue can be obtained from several sources, some critical aspects, such as the age of the animals, ovarian cyclicity, reproductive phase, and the types of ovarian structures, should be considered. Therefore, this review will focus on providing an update on the most current advances concerning the critical factors able to influence equine preantral follicle quality and quantity. Also, the in vivo strategies used to harvest equine ovarian tissue, the approaches to manipulating ovarian tissue post-harvesting, the techniques for processing ovarian tissue, and the classical approaches used to evaluate preantral follicles will be discussed.

Supportive techniques to investigate in vitro culture and cryopreservation efficiencies of equine ovarian tissue: A review.

During the reproductive lifespan of a female, only a limited quantity of oocytes are naturally ovulated; therefore, the mammalian ovary possesses a substantial population of preantral follicles available to be handled and explored in vitro. Hence, the manipulation of preantral follicles enclosed in ovarian tissue aims to recover a considerable population of oocytes of high-value animals for potential application in profitable assisted reproductive technologies (ARTs). For this purpose, the technique of preantral follicle in vitro culture (IVC) has been the most common research tool, achieving extraordinary results with offspring production in the mouse model. Although promising outcomes have been generated in livestock animals after IVC of preantral follicles, the quantity and quality of embryo production with those oocytes are still poor. In recent years, the mare has become an additional model for IVC studies due to remarkable similarities with women and livestock animals regarding in vivo and in vitro ovarian folliculogenesis. For a successful IVC system, several factors should be carefully considered to provide an optimum culture environment able to support the viability and growth of preantral follicles enclosed in ovarian tissue. The cryopreservation of the ovarian tissue is another important in vitro manipulation technique that has been used to preserve the reproductive potential in humans and, in the future, may be used in highly valuable domestic animals or endangered species. Several improvements in cryopreservation protocols are necessary to support the utilization of ovarian tissue of different species in follow-up ARTs (e.g., ovarian fragment transplantation). This review aims to provide an update on the most current advances regarding supportive in vitro techniques used in equids to evaluate and manipulate preantral follicles and ovarian tissue, as well as methodological approaches used during IVC and cryopreservation techniques.

The mule (Equus mulus) as a recipient of horse (Equus caballus) embryos: Comparative aspects of early pregnancy with mares.

The aim of this study was to compare the embryonic and early fetal development of horse embryos between recipient mules and mares from day 10-60 of pregnancy, in addition to hormonal (eCG and progesterone), ovarian, and uterine characteristics for approximately 4 months. Embryo donor mares (n = 5) and two groups of recipients (acyclic mules, n = 7; cyclic mares, n = 7) were used. Donor mares were monitored daily by transrectal ultrasonography and inseminated using fresh semen. Cyclic recipient mares were synchronized with the donor's ovulation using PGF2α and deslorelin acetate. Mules were prepared for the embryo transfers with estrogen and progestagen. Embryo collection and transfer were performed 8 days after ovulation of the donor mares. Pregnancy diagnosis with ultrasonography began 1 day after embryo transfer. After pregnancy confirmation, the recipient mules received long-acting progesterone once weekly for at least 120 days. The first day of detection (day 10) of an embryonic vesicle (EV) was similar between mules and mares. A period of extensive intrauterine mobility of the embryonic vesicle was observed similarly in mules and mares from days 10-17. The day of fixation of the EV in mules tended to be 1-day earlier than in mares; however, the diameter and growth rate of the EV did not differ between the two species. The embryo proper was first detected at day 20, and the crown-rump, width, and diameter were similar between the two recipient types. The heartbeat and allantoic sac tended to be detected 1 day later in mules than in mares, while the umbilical cord was first observed around day 40 in both species. Besides the expected differences found in ovarian aspects and eCG production, similar endometrial diameter, uterine tone and echotexture, and progesterone levels were seen between the two types of recipients. In conclusion, striking ultrasound similarities in equine embryo and fetal development, and uterine characteristics were seen between mules and mares used as recipients of horse embryos.

Deficiency in proliferative, angiogenic, and LH receptors in the follicle wall: implications of season toward the anovulatory condition.

This study aimed to gain insight on the effect of different seasons of the year on the expression pattern of growth factor and hormone receptors involved in follicle development. A novel follicle wall biopsy technique was used to collect in vivo follicle wall layers (ie, granulosa, theca interna, and theca externa) and follicular fluid samples from growing dominant follicles, simultaneously and repeatedly, using the same mares during the spring anovulatory (SAN), spring ovulatory (SOV), summer (SU), and fall ovulatory (FOV) seasons. The immunofluorescent expression patterns of epidermal growth factor receptor (EGFR), Ki-67, vascular endothelial growth factor receptor (VEGFR), and LH receptor (LHR) were evaluated in each follicle wall layer, in addition to intrafollicular estradiol and nitric oxide (NO). Proliferative proteins (EGFR and Ki-67) were highly (P < 0.05-P < 0.001) expressed during the SOV season compared with the SAN and FOV seasons. Lower (P < 0.05-P < 0.001) expression of both proteins was observed during SU compared with the SOV season. The expression of VEGFR was greater (P < 0.05-P < 0.01) in the theca interna of dominant follicles during the SOV season compared with the SAN and SU seasons. Similarly, in the overall quantification, the VEGFR expression was greater (P < 0.001) during the SOV season compared with the SU and FOV seasons. A higher (P < 0.05) LHR expression was detected in the theca interna during the SOV season than the SAN season. Furthermore, a higher (P < 0.05-P < 0.001) expression of LHR was observed in the granulosa, theca interna, and in the overall quantification during the SOV season compared with the SU and FOV seasons. Intrafollicular NO concentration did not differ (P > 0.05) among different seasons of the year. The intrafollicular estradiol concentration was higher (P < 0.05) during the SU compared with the SAN season and higher (P < 0.05) during the FOV season compared with the SAN and SOV seasons. In conclusion, the synergistic effect of lower expression of proliferative protein, angiogenic, and LH receptors in at least some of the layers of the follicle wall seems to trigger dominant follicles toward the anovulation process during the spring and fall transitional seasons.

Relationship of vascular perfusion of the wall of the preovulatory follicle to in vitro fertilisation and embryo development in heifers.

The effect of the extent of vascular perfusion of the wall of the preovulatory follicle on in vitro cleavage rate of the recovered oocyte and embryo development to >8 cells was studied in 52 heifers. Heifers received a luteolytic dose of prostaglandin F2alpha (PGF2alpha) when the largest follicle was > or =11 mm. An ovulation-inducing injection of GnRH was given 36 h later (hour 0), and collection of follicular fluid and the oocyte was done at hour 26. Vascular perfusion of the follicular wall was assessed by colour Doppler ultrasonography at hours 0 and 26. Each of the recovered oocytes (41/52; 79%) was mature (extruded polar body). Cleavage and embryo development were assessed at 48 h and 120 h respectively, after in vitro fertilisation (IVF). The percentage of cleaved oocytes and >8 cell embryos was 80% (31/39) and 55% (17/31) respectively. Vascular perfusion of the follicular wall was greater (lower pulsatility index; P<0.001) for follicles that produced cleaved versus non-cleaved oocytes and greater (P<0.04) for follicles that produced >8 cell versus < or =8 cell embryos. Percentage of follicular wall with Doppler signals of blood flow was greater (P<0.001) for >8 cell versus < or =8 cell embryos. Follicular-fluid concentration of free IGF1 was lower for cleaved oocytes (P<0.001) and >8 cell embryos (P<0.05), and oestradiol was lower (P<0.05) for >8 cell embryos. Results supported the hypothesis that greater vascular perfusion of the wall of the preovulatory follicle was positively associated with IVF and embryo development.

Age-related dynamics of follicles and hormones during an induced ovulatory follicular wave in mares.

An ovulatory follicular wave was induced by ablation of follicles > or =6mm and treatment with prostaglandin F2alpha (PGF) on Day 10 (ovulation=Day 0). Follicle and hormone dynamics of the induced waves were compared among three age groups: young (5-6 y, n=14 waves), intermediate (10-14 y, n=16), and old (> or =18, n=15). During the common-growth phase of the induced wave (Days 12-17), diameter of the future ovulatory follicle was not different among ages, but the young group had more (P<0.05) follicles that reached > or =10mm. The number was correlated (r=+0.7; P<0.0001) within mares between consecutive interovulatory intervals, indicating repeatability. Concentrations of LH increased in all age groups during Days 12-17, but were greatest (P<0.002) in the young group and continued to be greater (P<0.0001) throughout the ovulatory LH surge. During several days before Day-1, there were no age-related effects on systemic estradiol concentrations, diameter of the preovulatory follicle, or B-mode echotexture or color-Doppler signals of blood flow in the follicle wall. Interpretations were: (1) greater number of follicles in the young group reflected a greater follicle reserve, (2) greater LH concentrations throughout the ovulatory surge in the young group reflected a more positive response to an extraovarian/environmental influence after removal of the negative effect of progesterone, and (3) lower LH concentrations in the older groups were adequate for the preovulatory changes in the follicle.