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.

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.

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.

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.

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.

Pre-ovulatory follicle affects corpus luteum diameter, blood flow, and progesterone production in mares.

Color Doppler ultrasonography was used to study the temporal relationships between pre-ovulatory follicle (POF) and corpus luteum (CL) diameter and blood flow, with systemic progesterone (P4) concentration during two transitional ovulatory seasons in mares. Variables of POF and CL/P4 were evaluated for 6days before and 17days after ovulation, respectively. Evaluations were performed during two consecutive estrous cycles in spring and fall seasons, and during the last estrous cycle of the season. There were significant correlations among POF and CL variables, and P4 concentration that ranged from 0.24 to 0.95, and among the ratios of different variables that ranged from 0.39 to 0.92. There were linear regressions (P<0.01-0.001) for all comparisons among different variables. The POF diameter before the first ovulation of the season was larger (P<0.05), and POF vascularity was less (P<0.05), than in the last estrous cycle during the season. The CL blood flow was less (P<0.01) during the last compared with first pre-ovulatory period of the season. The POF diameters were positively correlated (r=0.67) during the two pre-ovulatory periods of spring and fall. Results provide evidence that the POF affects CL diameter and blood flow, and subsequently P4 production, and that POF diameter is repeatable within the same individual during different seasons.

Effects of FSH addition to an enriched medium containing insulin and EGF after long-term culture on functionality of equine ovarian biopsy tissue.

The effect of FSH supplementation on an enriched cultured medium containing insulin (10 ng/mL) and EGF (50 ng/mL) was investigated on in vitro culture of equine ovarian biopsy tissue. Ovarian tissue fragments were collected from mares (n = 10) and distributed in the following treatments: noncultured control, cultured control, and cultured + FSH. Both treated groups were cultured for 7 or 15 days. The end points evaluated were: follicular morphology, estradiol levels in the culture medium, fluorescence intensity for TUNEL, EGFR and Ki-67 detection, and gene expression of GDF-9, BMP-15, and Cyclin-D2 in the ovarian tissue. After seven days of culture, medium supplemented with FSH had a similar (P > 0.05) percentage of morphologically normal follicles compared to the noncultured control group. Estradiol levels increased (P < 0.05) from Day 7 to Day 15 of culture for both treated groups. No difference (P > 0.05) was observed for TUNEL and EGFR intensity between the noncultured control group and the treated groups after 15 days of culture. Ki-67 intensity did not differ (P > 0.05) between treated groups after 15 days of culture, but decreased (P < 0.05) when compared with the noncultured control group. Similar (P > 0.05) mRNA expression for GDF-9, BMP-15, and Cyclin-D2 was observed among all treatments after 15 days of culture. In conclusion, an enriched medium supplemented or not with FSH was able to maintain the functionality of equine ovarian biopsy tissue after a long-term in vitro culture.

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.

Glucocorticoid metabolism in equine follicles and oocytes.

The objective of this study was to determine whether (1) systemic and intrafollicular cortisol concentrations in horses are directly related and (2) supraphysiological levels of glucocorticoids affect in vitro maturation (IVM) rates of oocytes. Specifically, we studied the (1) changes in the intrafollicular cortisol and progesterone in context with granulosa cell gene expression during maturation of equine follicles (from 5-9 mm, 10-14 mm, 15-19 mm, 20-24 mm, and ≥25 mm in diameter) and (2) effects of cortisol supplementation on IVM rates and gene expression of equine cumulus-oocyte complexes (COCs). For these purposes, follicular fluid, granulosa cells, and COCs were collected from 12 mares (mean age 8.6 ± 0.5 yr) by transvaginal aspiration. Cortisol and progesterone concentrations in follicular fluid from follicles ≥25 mm were greater (P < 0.05) than in all other follicle classes and were positively correlated (r = 0.8; P < 0.001). Plasma concentrations of cortisol and progesterone did not differ before and after follicle aspiration (P > 0.05). In granulosa cells, gene expression of NR3C1, HSD11B1, HSD11B2, and CYP21A2 did not differ (P > 0.05) among different follicle classes. Maturation rates were similar (P > 0.05) among groups, regardless of the cortisol concentration in the IVM medium. In cumulus cells, messenger RNA expression of genes involved in glucocorticoid mechanism and apoptosis was either increased (NR3C1 and BCL2) or decreased (HSD11B2) by treatment (P < 0.01). In oocytes, gene expression of maturation markers (BMP15 and GDF9) was affected (P < 0.001) by cortisol treatment. This study demonstrates the involvement of glucocorticoids in follicle and oocyte maturation and cortisol modulation by HSD11B2 in equine COCs. Our data provide further information for understanding the normal ovarian endocrine physiology which might in turn also help improve equine assisted reproduction techniques.

Changes in intrafollicular concentrations of free IGF-1, activin A, inhibin A, VEGF, estradiol, and prolactin before ovulation in mares.

Changes in intrafollicular growth factors and hormones were evaluated in vivo in postdeviation and impending ovulation follicles. Mares (n = 30) were randomly assigned to five experimental groups based on target diameters of 25, 30, 35, 40 mm, and impending signs of ovulation. Furthermore, data belonging to two or more proximal diameter groups that were not different were combined and regrouped for each factor separately. Follicular fluid-free insulin-like growth factor 1 was highest (P < 0.003) in 35-mm follicles, followed by the 40-mm and impending ovulation follicle group, and the 25- to 30-mm follicle group. However, concentrations of insulin-like growth factor binding protein 2 in follicular fluid did not differ (P > 0.05) among groups. Additionally, follicular fluid activin A tended (P < 0.06) to be higher in impending ovulation follicles when compared with the 25- to 40-mm follicle group. Concentrations of intrafollicular estradiol were higher (P < 0.0001) in 40-mm and impending ovulation follicles than in the other follicle groups. Follicular fluid concentrations of inhibin A and vascular endothelial growth factor were lower (P < 0.05) in the 40-mm and the impending ovulation follicle group when compared with the 25- to 35-mm follicle group. Systemic and intrafollicular prolactin levels were lower (P < 0.05) in the impending ovulation group when compared with the 25- to 40-mm follicle group. Prolactin concentrations were higher (P < 0.05) in the follicular fluid than in the plasma. The novel findings of this study, a decrease in intrafollicular-free insulin-like growth factor 1, inhibin A, vascular endothelial growth factor, and prolactin during the final stages of follicular growth, document for the first time the occurrence of dynamic changes among intrafollicular factors and hormones during the stages of follicle dominance and as ovulation approaches.

FSH supplementation to culture medium is beneficial for activation and survival of preantral follicles enclosed in equine ovarian tissue.

This study investigated the effect of adding different concentrations of bovine recombinant follicle-stimulating hormone on the IVC of equine preantral follicles enclosed in ovarian tissue fragments. Randomized ovarian fragments were fixed immediately (fresh noncultured control) or cultured for 1 or 7 days in α-MEM(+) supplemented with 0, 10, 50, and 100 ng/mL FSH and subsequently analyzed by classical histology. Culture media collected on Day 1 or Day 7 and were analyzed for steroids (estradiol and progesterone) and reactive oxygen species (ROS). After Day 1 and Day 7 of culture, 50-ng/mL FSH treatment had a greater (P < 0.05) percentage of morphologically normal follicles when compared to the other groups, except the 10-ng/mL FSH treatment at Day 1 of culture. The percentage of developing follicles (transition, primary, and secondary), and follicular and oocyte diameters were higher (P < 0.05) in the 50-ng/mL FSH treatment compared to the other groups after Day 7 of culture. Furthermore, estradiol secretion and ROS production were maintained (P > 0.05) throughout the culture in the 50-ng/mL FSH treatment. In conclusion, the addition of 50 ng/mL of FSH promoted activation of primordial follicles to developing follicles, improved survival of preantral follicles, and maintained estradiol and ROS production of equine ovarian tissue after 7 days of culture.

Long-term in vitro culture of bovine preantral follicles: Effect of base medium and medium replacement methods.

Two culture media and replacement methods were compared during long-term in vitro culture of secondary follicles of cattle using α-MEM(+) or TCM-199(+) as base media. The medium replacement methods were: Conventional - removal and subsequent addition of the same amount (60µl) in a 100µl aliquot (MEM-C and TCM-C), and Small Supplementation - addition of 5µl of fresh medium to an initial small aliquot (50µl), resulting in a final volume of 125µl on the last day of culture (MEM-S and TCM-S). A total of 207 secondary follicles were cultured individually for 32 days at 38.5°C in 5% CO2 and medium replacement was performed every other day. The MEM-S treatment resulted in a larger (P<0.01) follicular diameter, greater (P<0.02) growth rate, greater (P<0.02) antrum formation, as well as greater (P<0.0001) estradiol concentrations when compared with the MEM-C treatment. The medium change methods did not affect (P>0.05) the follicular and estradiol end points for TCM-199(+). The expression of the FSHR gene was greater (P<0.03) with the TCM-C than TCM-S treatment, while the relative amounts of mRNA for IGF1 was greater (P<0.02) with MEM-S than TCM-S treatments and for VEGF was greater (P<0.02) with MEM-C than TCM-C treatment. In conclusion, the type of base medium and the effect of periodic addition of medium differentially affected follicle development, estradiol production, and gene expression. Furthermore, α-MEM(+) can be used to replace TCM-199(+) for culture of preantral follicles of cattle if progressive addition of medium is used for medium change.

In vitro development of bovine secondary follicles in two- and three-dimensional culture systems using vascular endothelial growth factor, insulin-like growth factor-1, and growth hormone.

The aim of this study was to evaluate the development and estradiol production of isolated bovine secondary follicles in two-dimensional (2D, experiment 1) and three-dimensional (3D using alginate, experiment 2) long-term culture systems in the absence (control group; only α-MEM(+)) or presence of vascular endothelial growth factor (VEGF), insulin-like growth factor-1, or GH alone, or a combination of all. A total of 363 isolated secondary follicles were cultured individually for 32 days at 38.5 °C in 5% CO2 in a humidified incubator with addition of medium (5 µL) every other day. In 2D culture system, follicular growth and antrum formation rates were higher (P < 0.05) in VEGF treatment compared with the other treatments. In 3D culture system, only estradiol concentration was greater (P < 0.05) in the GH than in the control group, whereas the other end points were similar (P > 0.05). In summary, this study demonstrated that the benefits of using a certain type of medium supplement depended on the culture system (2D vs. 3D). Vascular endothelial growth factor was an effective supplement for the in vitro culture of bovine secondary follicles when the 2D culture system was used, whereas GH only affected estradiol production using the 3D culture system. This study sheds light on advancements in methodology to facilitate subsequent studies on bovine preantral follicle development.

In vitro culture of equine preantral follicles obtained via the Biopsy Pick-Up method.

The objective was to conduct a preliminary evaluation of the efficacy of two media for in vitro culture of equine preantral follicles. Ovarian cortical strips were obtained from mares (N = 10) via the Biopsy Pick-Up method during the breeding season. Ovarian tissue was immediately submitted to histological analysis (noncultured control; D0) or cultured in situ for 1 day (D1) or 7 days (D7) in either α-MEM or TCM-199 and submitted to histological analysis, generating five treatment groups: noncultured control, α-MEM:D1, TCM-199:D1, α-MEM:D7, and TCM-199:D7. Preantral follicles were evaluated for follicle class (primordial, transitional, primary, and secondary) and morphology (normal vs. abnormal). A total of 142 preantral follicles were analyzed in five replicates. No follicles were observed in the TCM-199:D7 treatment group. The proportion of primordial follicles was higher (P < 0.03) in the control compared to the α-MEM:D7 treatment group. The proportion of primary follicles was higher (P < 0.04) in the α-MEM:D7 treatment group compared to the control. The proportion of developing follicles (transitional, primary, and secondary) was higher (P < 0.03) in the α-MEM:D7 treatment group compared to the control group. There was a greater (P < 0.004) percentage of morphologically normal developing follicles in the α-MEM:D1 treatment group compared to the TCM-199:D1 treatment group. Overall, the percentage of morphologically normal follicles was higher in the control group (72%; P < 0.02) and α-MEM:D1 group (84%; P < 0.0001) compared to the α-MEM:D7 (27%) treatment group. Mean follicle diameter was greater (P < 0.04) in the α-MEM:D7 treatment group (40.6 ± 1.1 µm) compared to the control group (37.3 ± 0.7 µm). Mean oocyte diameter was greater in the α-MEM:D1 (31.0 ± 0.7 µm; P < 0.006), TCM-199:D1 (30.7 ± 1.8 µm; P < 0.006), and α-MEM:D7 (33.2 ± 1.8 µm; P < 0.006) treatment groups compared to the control group (27.4 ± 0.9 µm). In conclusion, based on these preliminary data, in vitro culture of equine ovarian fragments obtained in vivo via the Biopsy Pick-Up method promoted preantral follicle development and follicle and oocyte growth in α-MEM for 7 days, with some follicles remaining morphologically normal throughout the culture period.

Equine preantral follicles obtained via the Biopsy Pick-Up method: histological evaluation and validation of a mechanical isolation technique.

The aims of this study in mares were to: (1) compare preantral follicle parameters between in vitro Biopsy Pick-Up (BPU) and scalpel blade collection methods and between histological and mechanical isolation processing (experiment 1); (2) histologically evaluate preantral follicles (experiment 2); and (3) compare histological analysis with a previously established mechanical isolation technique using a tissue chopper (experiment 3) for ovarian cortical fragments obtained in vivo using a BPU instrument. In experiment 1, preantral follicles were analyzed (N = 220; 90% primordial and 10% primary). Proportions of primordial and primary follicles did not differ (P > 0.05) between tissue collection (BPU vs. scalpel blade dissection) or processing (mechanical isolation vs. histology) methods. Follicle viability and morphology rates were similar (P > 0.05) between tissue collection methods, but mechanical isolation produced more (P < 0.05) morphologically normal follicles than histology. For experiment 2, preantral follicles (N = 332) were analyzed and primordial and transitional (combined) follicles and oocytes were 36.3 ± 0.3 and 26.1 ± 0.3 µm in diameter, respectively, and primary follicles and oocytes averaged 42.9 ± 1.8 and 31.8 ± 2.1 µm. For experiment 3 (188 preantral follicles), within the same animals, the proportion of primordial versus primary follicles was higher (P < 0.03) for histological analysis (98%) compared to tissue chopper analysis (94%), and number of follicles per mg of tissue was not affected (P > 0.05) by processing methods. In conclusion, most parameters evaluated for preantral follicles were similar between histological and tissue chopper processing techniques; hence, mechanical isolation efficiently dissociated equine preantral follicles from the ovarian cortex. Therefore, the tissue chopper could be used to isolate large numbers of morphologically normal equine preantral follicles for cryopreservation and/or in vitro culture.

Quantification, morphology, and viability of equine preantral follicles obtained via the Biopsy Pick-Up method.

A Biopsy Pick-Up (BPU) method was tested to determine the feasibility of retrieving preantral follicles from mare ovaries in vivo. A total of 33 ovarian biopsy procedures were performed on 18 mares during the breeding season. Mares were 5 to 21 years old and biopsies were performed during the estrous and/or diestrous phase, as confirmed by transrectal ultrasonography. Follicles were mechanically isolated using a tissue chopper, counted, and classified as normal or abnormal and primordial or primary. Viability of isolated follicles was determined by Trypan Blue dye. A total of 256 biopsy attempts were made resulting in 185 successful tissue sample collections (72% success rate). The mean weight of ovarian tissue collected per procedure was 25.0 ± 1.6 mg. Overall, 620 preantral follicles were collected and isolated (95% primordial and 5% primary). The mean (±SEM) number of follicles isolated per biopsy procedure was 18.8 ± 1.9. Primordial and primary follicles had an average diameter of 31.3 ± 6.2 and 41.1 ± 6.6 µm, respectively. Viability rate was higher (P < 0.001) for primordial follicles (91%) compared with primary follicles (50%). Primordial follicles tended (P < 0.06) to have a higher rate of morphological normality (96%) compared with primary follicles (80%). The total number of follicles isolated, amount of tissue harvested, and number of follicles per mg of tissue did not differ (P > 0.05) according to phase of the estrous cycle. Younger mares (5 to 7 years old) had more (P < 0.05) follicles isolated per procedure than older mares (14 to 21 years old). The length of the interovulatory interval was not affected (P > 0.05) by any biopsy procedure, and there were no adverse effects on cyclicity or general reproductive health. In conclusion, the BPU method provided large numbers of normal and viable preantral follicles for the study of early follicular development in mares. The BPU method might be used in the future to obtain preantral follicles for in vitro culture to enable the use of numerous oocytes present within the equine ovary. This could allow for the preservation of genetic material or large-scale embryo production.

Short-term feed restriction decreases the systemic and intrafollicular concentrations of leptin and increases the vascularity of the preovulatory follicle in mares.

The objective of this study was to evaluate the effect of short-term feed restriction on characteristics of the preovulatory follicle and on concentrations of systemic hormones (leptin, follicle-stimulating hormone [FSH], luteinizing hormone [LH]) and follicular fluid hormones and growth factors (leptin, estradiol, inhibin-A, activin-A, free insulin-like growth factor-1 [IGF1], insulin-like growth factor binding protein 2 [IGFBP2], vascular endothelial growth factor [VEGF]). Mares were submitted to a short-term (48 h) feed restriction when the expected ovulatory follicle was > or =27 mm (Hour 0) or served as controls (n=8/group). No effect of short-term feed restriction was detected for systemic concentrations of FSH and LH and for intrafollicular concentrations of estradiol, activin-A, free IGF1, and IGFBP2. Restricted mares had decreased systemic concentrations of leptin at Hour 24 (approached significance) and at Hours 36 and 48 (P<0.04). Follicular fluid of restricted mares at Hour 48 had lower (P<0.02) concentration of leptin and a tendency (P<0.1) for greater concentrations of inhibin-A and VEGF. The percentage of wall of the preovulatory follicle with color-Doppler signals of blood flow at Hour 48 was greater (P<0.04) in the restricted group. Intrafollicular concentration of leptin (combined groups) was positively correlated with score for body condition (r=+0.60; P<0.002) and negatively correlated with the percentage of the follicle wall with blood-flow signals (r=-0.60; P<0.02). Our favored interpretation is that the preovulatory follicle seems to compensate for a nutritional deficiency by increasing the blood flow in the follicle wall.

Follicle suppression of circulating follicle-stimulating hormone and luteinizing hormone before versus after emergence of the ovulatory wave in mares.

The effect of the ovarian follicles on plasma concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) before versus after the expected emergence of the ovulatory follicular wave was studied on Days 0 to 18 (Day 0=ovulation) in four groups of mares (n=6/group). In addition to a control group, all follicles >/=6mm in diameter were ablated on Days 0.5, 6.5, or 12.5 in a herd of mares with reported emergence at 6mm of the future ovulatory follicle on mean Day 10.5. Concentrations of FSH were not different between the Day-0.5 or Day-6.5 ablation groups and the corresponding controls. However, ablation on Day 12.5 resulted in an immediate FSH increase (group-by-day interaction, P<0.003). For LH, ablation on Day 0.5 resulted in an interaction (P<0.02), partially from lower (P<0.05) concentrations on each of Days 15.5 to 18.0 than that in the controls, whereas ablation on Days 6.5 or 12.5 did not result in a significant group effect or interaction. Testosterone concentration, but not progesterone or estradiol concentration, was lower (P<0.04) on Day 2 in the Day-0.5 ablation group than that in the controls. We inferred that follicles did not contain adequate FSH suppressors on Days 0.5 and 6.5 and that they were present only in the Day-12.5 ablation group or after the expected emergence of the ovulatory wave. The hypothesis of an association between low postovulatory concentrations of an ovarian steroid and low concentrations of LH after Day 15 was supported.

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.