Medium Follicles Research Articles

Proteome and metabolomic profile of Mongolian horse follicular fluid during follicle development

During follicular development, changes in the composition of the follicular fluid are synchronized with the development of oocytes. Our aim was to screen the key factors affecting oocyte maturation and optimize the in vitro culture protocol by understanding the changes of proteins and metabolites in follicular fluid. Follicles are divided into three groups according to their diameter (small follicle fluid (SFF): 10 mm < d < 20 mm; medium follicle fluid (MFF): 20 mm < d < 30 mm; large follicle fluid (LFF): 30 mm < d). Proteins and metabolites from the follicular fluid were analyzed by mass spectrometry. The results showed that: in LFF vs MFF, 20 differential abundant protein (DAP) and 88 differential abundant metabolites (DAM) were screened out; In SFF vs MFF, 3 DAPs and 65 DAMs were screened out; In MFF vs SFF, 24 DAPs and 35 DAMs were screened out. The analysis of differential proteins and metabolites showed that glycerophosphate hydrolysis decreased during follicular development, and proteins played a major role in metabolism and binding. In addition, DAMs and DAPs are co-enriched in the “linoleic acid metabolism” pathway. Combinatorial analysis reveals the dynamic profile of follicular fluid during follicular development and provides fundation for further exploring the function of follicular fluid in Mongolian horse.

P-771 Does follicle size impact effect of follicular flushing on oocyte retrieval rate and embryology outcomes?

Abstract Study question Is the effect of follicular flushing on oocyte retrieval rate (RR) and embryology outcome influenced by follicular size? Summary answer Flushing improved RR in discordant growth with smaller(8-12 mm) follicles and larger(&amp;gt;22mm) follicle. Flushing did not impact embryological outcomes adversely. It increased top quality embryo What is known already Follicular flushing is done in poor responder for improving retrieval rate. There are very few studies in normal or hyper responders. Studies show controversial results of flushing, some showing benefits, while others, a detrimental effect because of increased intra-follicular pressure, increased procedure time, and change in paracrine milieu of oocytes by dilution with flushing medium No study has addressed whether increased retrieval rates with flushing are dependent on follicular size and indirectly maturity of oocyte. Most studies flushed with fixed volume of 1-2ml as size of follicle was not taken into account, making volume inadequate for completely flushing larger follicles. Study design, size, duration Prospective cohort study conducted from September 2022 to August 2023. Group 1: Study group- follicles were flushed after aspiration. Total patients were 120. They were divided into 3 groups according to follicular size and fluid volume aspirated with sample size calculated as A:306 small follicles 8-12 mm(&amp;lt;1.0ml), B:467 medium follicles 13-22 mm (1.0-6.0ml) C:102 large follicle &amp;gt; 22 mm (&amp;gt;6.0 ml). Group 2- Control group where follicles aspirated without flushing in 280 patients, 2572 follicles. Participants/materials, setting, methods Ethical approval taken. Inclusion - patients 21-40years of age undergoing oocyte retrieval for IVF/ICSI cycle with antagonist protocol Exclusion of oocyte of indeterminate origin ie one aspirate have two oocytes Group1 After aspiration of follicle with double lumen oocyte retrieval needle, follicle flushed with media volume equivalent to follicular volume. Group2 only follicular aspiration and volume measured Analysis done using SPSS-25 version. Primary outcome retrieval rate(RR) of oocytes. Secondary outcome: fertilization rate, percentage of top quality embryos(TQE)on day3&amp;5, blastocyst rate. Main results and the role of chance Flushing follicle significantly increased RR in smaller follicles(8-12mm) compared to controls 159/311 (51.12%)vs 176/780 (22.56%) p = &amp;lt;0.001 and also in larger follicles(&amp;gt;6ml) 76/109 (69.72%)vs 116/219 (52.96%) p = &amp;lt;0.01 No difference seen in optimum size follicles (13-22mm) 337/470 (71.7%)vs 1099/1573(69.86%) p = 0.44. Overall, there was statistically significant increase in RR with flushing 567/890 (63.7%)vs 1391/2572 (54.08%) p = &amp;lt;0.001. Significantly more time was taken for flushing vs control groups 16.50vs 12.15 minutes p = &amp;lt;0.001. Secondary outcomes like fertilization rate was not different with flushing 476/567 (83.95±15.01%)vs 1125/1391(80.88±19.01 %) p = 0.21. Percentage of TQE (day 3) were significantly more in flushing group 321/476 (67.43±25.41%)vs control 642/1125 (57.07±25.94%)p = &amp;lt;0.01. TQE (day 5) were significantly more in flushing group 142/476 (29.83±11.02)vs control 251/1125 (22.31±12.11%)p=0.04. Blastocyst rate remained similar in both groups 196/476 (41.18±16.73 %) vs 403/1125 ( 35.82±13.90 %) p = 0.12. Subgroup analysis showed that younger women (&amp;lt;35yrs)had a significantly higher RR with flushing 66.33±16.29%vs controls 57.88±13.68% (p = &amp;lt;0.01), compared to older women &amp;gt;35 years 60.29±16.37%vs 56.35±17.30% (p = 0.38). Women with higher AMH had better RR with flushing 63.71±15.11%vs controls 56.50±14.33 (p = 0.01) compared to women with lower AMH (&amp;lt;1 ng/ml) where flushing made no difference 63.47±19.59%vs 60.62±14.93(p = 0.79). Normal and hyper responders had significantly higher RR with flushing 66.16±15.11vs controls 57.53±13.85%(p = &amp;lt;0.001) compared to poor responders (&amp;lt;5 oocytes) 51.07±18.07vs 56.24±27.47(p = 0.55). Limitations, reasons for caution Although RR was better with flushing in small and large follicles we could not translate the advantage to pregnancy rate(PR) PR could not be calculated as some patients wanted 2 embryos transferred. Since patient had one each embryos from small and optimum follicles both were transferred. Further RCT required. Wider implications of the findings Practice of follicular flushing needs re-evaluation Women with discordant growth should be taken for flushing as it increases RR in small and large follicles Role of flushing should be widened beyond poor responders to younger women, with normal or hyper response increasing oocyte yield which may increase cumulative pregnancy rate Trial registration number not applicable

The impact of cumulus cell viability and pre-culture with the healthy cell mass on brilliant cresyl blue (BCB) staining assessment and meiotic competence of suboptimal porcine oocytes

Objectives of the present study were to investigate the characteristics including glucose-6-phosphate dehydrogenase activity, as determined by Brilliant Cresyl Blue (BCB) staining, of suboptimal porcine oocytes and to enhance the meiotic competence of those through pre-culture with cumulus cell masses (CCMs). Percentage of oocyte-cumulus complexes (OCCs) derived from small follicles (SF; <3 mm in diameter) containing the oocytes that were assessed as BCB-negative (BCB-) was significantly higher than those derived from medium follicles (MF; 3–6 mm in diameter). Degrees of dead cumulus cells were significantly higher in OCCs containing BCB- oocytes, regardless of the origin of OCCs (MF vs. SF), than those containing BCB-positive (BCB+) ones. Exposing OCCs containing BCB+ oocytes to the apoptosis inducer, carbonyl cyanide m-chlorophenylhydrazone, for 20 h significantly induced the transition to BCB- and meiotic progression of exposed OCCs were significantly reduced in both SF and MF derived ones. Transit of BCB- oocytes to BCB+ was induced when OCCs were pre-cultured with CCMs of MF derived OCCs containing BCB+ oocytes for 20 h before IVM. This pre-culture also significantly increased the meiotic competence of BCB- oocytes, particularly in SF derived ones. However, reactive oxygen species levels were significantly higher in BCB+ oocytes as compared with BCB- ones, regardless of pre-culture with CCMs, whereas no significant differences were found in the ATP contents among the treatment groups. In conclusion, the BCB result of oocytes could be regulated by the healthy status and content of surrounding cumulus cells and the meiotic competence of suboptimal BCB- porcine oocytes is improved by pre-culture with healthy CCMs.

The effectiveness of different hormone protocols for improving ovarian function before ovum pick-up in crossbred Japanese black cattle.

Producing and transferring embryos in vitro are profitable for enhancing premium beef genetics. Reducing costs and enhancing the effectiveness of hormone protocols before ovum pick-up (OPU) yield advantages. This study aimed to confirm that estradiol benzoate (EB) treatment resulted in more medium- and large-sized follicles before OPU and of higher oocyte quality after OPU than non-hormonal treatments, comparable to those undergoing gonadotropin-releasing hormone (GnRH) with follicle-stimulating hormone (FSH) plus prostaglandin F2α (PGF2α) and progesterone-releasing controlled internal drug release (CIDR) or EB with progesterone-releasing CIDR hormonal treatments. 30 crossbred Japanese black cows were divided into five equal groups, which were either untreated or treated with different hormone protocols before OPU. Group 1 (cows in estrus) and group 2 (cows in diestrus) were the untreated controls. Cows in group 3 were treated with GnRH + FSH + CIDR + PGF2α. Cows in group 4 received EB, and those in group 5 received EB + CIDR + PGF2α. After administering the protocols, all cow follicles were examined through ultrasonography and categorized by size. Subsequently, all cows underwent OPU, and the oocytes were collected and graded from A to D according to standard criteria. Group 3 presented the highest large follicle numbers, and groups 3-5 had more medium follicle numbers, not different among groups but they had this parameter more than those of control groups 1 and 2. Moreover, groups 3-5 did not differ in combined grades A + B oocytes (good-quality oocytes). Groups 3 and 4 had more A + B oocytes than control groups 1 and 2, whereas group 5 was not different in this parameter from group 1. Among the three hormone protocols, EB treatment proved the most cost-effective and efficient, yielding more high-quality oocytes compared to the non-treatment protocols. To reduce the limitations of EB use in the future, this study suggests researching natural EB phytoestrogens as alternative treatments for improving ovarian function before OPU in other cattle breeds.

Morphological Characteristics of Ovarian Tissues and Follicular Fluid Metabolites of Female Lambs and Ewes in Subtropics

Background: Oocyte and follicular fluid recovery and evaluation are a crucial step in assisted reproductive technologies. The aims of the current study were to evaluate the ovarian tissues and their resulting oocytes and follicular fluid of Awassi female lambs and ewes in subtropics of Saudi Arabia. Methods: Thirty reproductive systems of female lambs in addition to forty reproductive systems of ewes of follicular and luteal phases were collected. The ovarian tissues were evaluated through ovarian follicle numbers, oocytes recovery in addition to determination of follicular fluid (FF) metabolites. The ovarian follicles were categorized into small ( less than 3 mm), medium (3-5 mm) and large follicles ( greater than 5 mm). The medium and large ovarian follicles were aspirated for FF collection and oocytes recovery. The FF concentrations of total protein (g/dl), urea (mg/dl), glucose (mg/dl), total cholesterol (mg/dl) and triglycerides (mg/dl) were determined. Result: The weight of reproductive system and ovary were increased of ewes compared to female lambs. The number of small follicles was not differed among groups whereas the numbers of medium and large follicles increase in ewes groups. The highest number of medium follicles was found in ewes of follicular phase compared to other groups. Although the percentage of oocyte recovery increased in ewes compared to lambs, the oocytes quality was not differed among groups. The total protein values were high (P less than 0.05) in FF of ewes compared to that of female lambs. On the other, the values of urea and triglycerides were low (P less than 0.05) in FF of ewes compared to FF of female lambs. Moreover, the values of total protein, urea and triglycerides were comparable in FF of ewes during follicular or luteal phase. Additionally, there is slight increase of glucose and slight decrease of total cholesterol values in FF of ewes compared to FF of female lambs. In conclusion, follicles of ovarian tissues, oocyte recovery and quality and follicular fluid biochemistry of female lambs and ewes showed differences. The recovered oocytes and FF are available for assisted reproductive techniques.

MORPHOMETRIC CHARACTERIZATION OF THE STRUCTURAL COMPONENTS OF THE THYROID GLAND UNDER CONDITIONS OF MODELED THERMAL SKIN INJURY

The key organ of the peripheral endocrine system, the thyroid gland, shows the ability to adapt and restructure at diff erent levels of structural and cellular organization under the infl uence of various factors. The aim of the study was to determine morphometric changes in the parameters of the structural components of the thyroid gland. The experiment was modeled on 42 male laboratory mature white rats. The burn was modeled with copper plates heated in water at a temperature of 97-100 °C. The size of the aff ected area was 20 % of the animal’s body area, which, according to the current classifi cation, corresponds to a seconddegree burn. Changes in various parameters were studied by morphometric analysis of the obtained histologic sections. Statistical processing of digital data was carried out using the software «Excel» and «STATISTICA» 10.0 using parametric and nonparametric methods of data evaluation. Morphometrically, it was found that the organ contains follicles of three types – large, medium and small, and their quantitative ratio is 1.4: 4,7: 3,9. It was found that the average value of the area of a small follicle is (2342.5498.43) μm2, medium – (3722.53130.44) μm2, large – (5733.77203.67) μm2. The average value of thyrocyte height is (11.430.41) μm. The follicular- colloid index of the thyroid gland of intact animals is the highest for small follicles and is 1.940.08, for medium follicles – 1.81 0.04 and for large follicles – 1.730.05. The average value of the colloid accumulation index for small follicles is the lowest and is 2.600.12, for medium follicles – 4.18 0.19, and for large follicles – 6.270.29. At the light- optical level, on day 7 of the experiment, an increase in the number (1.58 times (p&lt;0.001) compared to the intact index) of large follicles was noted, which are located mainly on the periphery of the lobules and fi lled with a compacted colloid. A decrease in thyrocyte height (1.29 times (p&lt;0.001) compared to the normal value), a signifi cant decrease in the follicular- colloid index and an increase in the colloid accumulation index detected in the early toxemia stage indicate a decrease in functional activity in the organ, disruption of adaptive processes in it and the onset of destructive changes. At the later stages of burn injury (14 and 21 days of the experiment), against the background of a statistically signifi cant (p&lt;0.001) increase in endogenous intoxication, there is a development of deep destructive changes in thyrocytes, organ vessels, proliferation of connective tissue layers, impaired synthesis and secretion of hormones. These conditions are manifested by the dominance of large, overstretched follicles in the parenchyma of the thyroid gland, lined with fl at, degenerating thyrocytes, the height of which at day 21 is 3.31 times signifi cantly (p&lt;0.001) lower than the intact value; a statistically signifi cant decrease in the follicular- colloid index; a signifi cant increase in the colloid accumulation index, as well as a signifi cant deviation from the normal level of pituitary- thyroid system homones, which indicates organ hypofunction.

Effect of use and dosage of p-follicle-stimulating hormone for ovarian superstimulation before ovum pick-up and in vitro embryo production in pregnant Holstein heifers

The benefit of ovarian superstimulation using exogenous FSH before ovum pick-up (OPU) and in vitro embryo production (IVEP) has been the subject of conflicting results. The objective of the present study, therefore, was to evaluate the effect of use and dose of porcine FSH (p-FSH) before OPU/IVEP on ovarian response and embryo production in pregnant heifers. Pregnant Holstein heifers (n = 48) were assigned randomly to receive 0, 160, or 300 mg NIH-FSH-P1 in a crossover design. Ovum pick-up was performed at 49, 63, and 77 d of gestation with a 14 d "washout" between OPU sessions. Follicle ablation was performed on D 0 (p.m.) and p-FSH treatments, consisting of 4 decreasing dose injections administered 12 h apart, were initiated 36 h after follicle ablation (D2 a.m.). Heifers underwent OPU on D 5 (a.m.), 40 h after the last p-FSH treatment, and cumulus oocyte complexes (COC) were subjected to IVEP procedures. Differences between treatment groups were evaluated using generalized linear mixed models. There were quadratic effects of treatment on both number and percentage of small (<6 mm), medium (6-10 mm) and large (>10 mm) follicles. Number and percentage of medium follicles increased with increasing p-FSH dosages, although the magnitude of the change was greater between 0 and 160 mg, than between 160 and 300 mg of p-FSH. Total number of follicles, number of COC recovered and number of viable COC increased linearly with increasing p-FSH dose. Conversely, there was no evidence for an effect of p-FSH dose on COC recovery percentage nor the percentage of viable COC. Cleavage percentage, number of cleaved oocytes, blastocyst percentage, and number of blastocysts increased linearly with increasing p-FSH dose. In conclusion, use of p-FSH before OPU resulted in greater superstimulatory response and oocyte competence which in turn increased IVEP. Furthermore, these effects were dose dependent such that use of a greater dose of p-FSH up to 300 mg progressively increased embryo yield.

Evaluation of cortisol, thyroxine (T3) and triiodothyronine (T4) concentrations in preovulatory follicles in jennies.

Reference values of follicular fluid components at different stages of follicular development in jennies are unknown. It is important to estimate such values because the biochemical composition of follicular fluid provides a suitable microenvironment for oocyte development, growth and maturation. The experimental protocol was carried out in the Equine Reproduction Clinic of the FMVZ-UNAM and was approved by an Institutional Sub-committee for the Care and Use of Experimental Animals (SICUAE.MMVZ-2020/4-2). Seven healthy female donkeys “Mexican burro” with average body condition score of 3 on a scale of 1 to 5 (Svendsen et al. The professional Handbook of the Donkey. 2008), were monitored by transrectal ultrasonography through the Veterinary Digital Diagnostic Ultrasound System 5 MHz (Model, DUS 60 VET) with a multi-frequency linear transducer. The ovum pick up (OPU) by transvaginal aspiration (TVA) was performed in a total of 70 preovulatory follicles, 10 for each jenny using a real- time B- mode ultrasound vaginal 6.5 MHz (Sonovet 600) with curvilinear transducer and 12 gauge (12 G) needle. Follicular fluid components were measured and analyzed by Luminex Multiplex Assays at the Universidad Nacional Autónoma de México. The relationship between cortisol, thyroxine (T3) and triiodothyronine (T4) concentrations in preovulatory follicles and follicular size was assessed. The preovulatory follicles are classified in three groups accordingto the diameter: small (27-30 mm), medium (31-35 mm) and large (36-38 mm). Concentrations of cortisol were lower in the large follicles group (18.8 ng/ml) compared with small (721.44 ng/ml) and medium follicles (361 ng/ml). The concentrations of thyroxine were higher in the medium follicle group (49 ng/ml) than in the small (21ng/ml) and large follicle groups (15.9 ng/ml). Finally, the concentration of triiodothyronine was higher in the small follicles group (1043 ng/ml) followed by the medium (545 ng/ml) and the lowest concentration was in the large follicles group (22 ng/ml). It is concluded that thyroxine concentrations are lower than T4 concentrations in preovulatory follicles irrespective of size as reported for human follicular fluid (Wakim. et. al. Fertil Steril 1993; 59(6) 1187–1190). Furthermore, no relationship was found between preovulatory follicular size and thyroxine hormone concentrations. It has been suggested that cortisol and triiodothyronine concentrations are related to preovulatory follicle size. Cortisol concentrations are higher in preovulatory follicles that in subordinate follicles and cortisol is a mediator of apoptosis in granulosa cells, differentiation, development and maturation of tissues, specifically in the oocyte (Acosta & Miyamoto. Anim Reprod Sci 2004; 82:127-140, Scarlett. et al. Dom Anim Endocrinol 2016; 59:11-22). The role of cortisol within the different sizes of preovulatory follicles is unknown.

Seasonal variation of serum and intrafollicular concentrations of melatonin and estradiol in relation to follicular development

The main objective of this study was to compare the effect of season on serum and intrafollicular concentrations of melatonin (MT). The relationship between follicular size, MT and estradiol (E2) concentrations was also explored. Twelve mares were used. Aspiration of fluid from all follicles larger than 5 mm was carried out in each mare during the breeding season and during the anovulatory season. All aspirations were carried out between 7AM and 8AM, and a serum sample was obtained at the same time. Follicles were classified into small (≤ 15 mm); medium (16-30 mm); and large (> 30 mm). MT and E2 concentrations were measured by enzymatic immunoassay (Melatonin ELISA Mybiosource, MBS1603596; Estradiol ELISA DRG, EIA-2693). Analysis of Variance was used to evaluate the effects of season, fluid type (follicular vs serum) and follicle size. Intrafollicular concentrations of MT were higher than serum concentrations both in the reproductive season (127±1.41 vs 69.59±1.73 ng/L) and in the anovulatory season (118.5±2.93vs 115.45±3.06 ng/L). MT concentrations in serum were higher during the anovulatory season than during the reproductive season (p<0.05), but the opposite was the case for intrafollicular MT. During the ovulatory season MT was higher (p<0.05) in large follicles (141.39±2.13ng/L) than in medium (130.25±2.55 ng/L) and small follicles (109.36±2.57 ng/L). The difference between medium and small follicles was also significant(p<0.05). In contrast during the anovulatory season MT concentration was not different between small and medium follicles (123.43±3.79 vs 109.92±5.55 ng/mL). Intrafollicular levels of E2 were higher during the ovulatory season than during the anovulatory season in medium (211.89±20.4 vs 126.18±0.07) and small follicles (67.42±2.4 vs 14.78±1.06 ng/mL). E2 concentrations in large follicles during the ovulatory season was 4,312.83±65.8 ng/mL. The results of this study indicate that while serum MT concentrations were different between seasons due to the known negative correlation between pineal MT secretion and day length, this seasonal effect was not found in the case of intrafollicular MT concentrations. Also, in this study there was no correlation between serum and intrafollicular MT concentrations. Together, these findings suggest photoperiod-independent intrinsic follicular MT synthesis. E2 concentrations were correlated with follicle size in both ovulatory and anovulatory seasons but were always higher during the ovulatory season for a given size. During this season there was a positive correlation between intrafollicular MT and estradiol concentrations, thus suggesting that intrafollicular estradiol may stimulate intrafollicular melatonin synthesis. It is concluded that intrafollicular MT concentrations in the mare are at least in part of intrinsic origin and may be stimulated by estradiol.

Lipid metabolites, interleukin-6 and oxidative stress markers in follicular fluid and their association with serum concentrations in mares.

The application of trans-vaginal ovum pick up (OPU) and intracytoplasmic sperm injection (ICSI) is well established for commercial in vitro embryo production in horses. These assisted reproductive techniques are especially applied during the non-breeding season of the mare. However, little is known about how the health of the oocyte donor may affect the biochemical composition of the follicular fluid (FF) in small and medium-sized follicles routinely aspirated during OPU. This study aimed to investigate associations between systemic and FF concentrations of interleukin-6 (IL-6), total cholesterol, triglycerides, non-esterified fatty acids (NEFA), reactive oxygen metabolites (d-ROMs), biological antioxidant potential (BAP), and oxidative stress index (OSI) during the non-breeding season in mares. At the slaughterhouse, serum and FF of small (5-10mm in diameter), medium (> 10-20mm in diameter), and large (> 20-30mm in diameter) follicles were sampled from 12 healthy mares. There was a strong positive association (P < 0.01) between the concentration of IL-6 in serum and those measured in small (r = 0.846), medium (r = 0.999), and large (r = 0.996) follicles. Serum concentrations of NEFA were positively correlated (P < 0.05) with those measured in small (r = 0.726), medium (r = 0.720), and large (r = 0.974) follicles. Values of total cholesterol and OSI in serum and medium follicles were significantly associated (r = 0.736 and r = 0.696, respectively). The serum concentrations of all lipid metabolites were markedly higher than those measured in FF of small- and medium-sized follicles. Values of IL-6 and OSI did not change significantly between serum and all follicle classes (P ≥ 0.05). To conclude, changes in the blood composition associated with inflammation, oxidative stress, and disturbed lipid metabolism of mares may lead to an inadequate oocyte microenvironment, which could affect oocyte quality and the success rate of OPU/ICSI programs. Further research should indicate whether these changes may ultimately affect in vitro oocyte developmental capacity and subsequent embryo quality.

Impact of follicular size categories on oocyte quality at trigger day in young and advanced-age patients undergoing GnRH-ant therapy.

To study the effect of follicle sizes of different proportions on oocyte and embryo quality in young and advanced-age patients, and provide evidence for personalized protocol adjustment. This was a retrospective real-world data study including a total of 11,462 patients who had started their first in vitro fertilization cycle with a gonadotropin-releasing hormone antagonist (GnRH-ant) protocol during 2018-2021. We classified patients into groups according to the size of the dominant proportion of follicles on the human chorionic gonadotropin (hCG) trigger day: Large, Medium, Small, and Equal (containing equivalent proportions of all three size categories). The Cochran-Mantel-Haenszel test by different Anti-Mullerian Hormone (AMH) and antral follicle count (AFC) was used to compare factors such as the metaphase II (MII) oocyte rate, normal fertilization rate, and two pronuclei (2PN) cleavage rate between groups. General linear model (GLM) analysis was performed for inter-group comparison of the oocyte and embryo quality. In patients aged < 35 years and with AMH ≥ 1.2μg/L, the MII oocyte percentages in the Large and Medium groups were significantly higher than in the Small group (P < 0.001). The germinal vesicle (GV) oocyte and unavailable oocyte percentages in the Large and Medium groups were lower than in the Small group (P < 0.001). Among patients aged ≥ 35 years with AFC < 5 and AMH ≥ 1.2μg/L, the GV oocyte percentage in the Large group was significantly lower than in the Medium group (2.54% vs. 4.46%, P < 0.001). In patients < 35 years, the GLM demonstrated that the Large and Medium groups had positively impacted on the development of MII oocyte and live birth rate(LBR) of first embryo transfer(ET)(β>0, all P value < 0.05);and had less likely to develop into unavailable oocyte, degenerated oocyte, GV oocyte and MI oocyte rates relative to the Small group(β<0, all P value < 0.05). And among patients ≥ 35 years, the Medium group had positively impacted on the development of MII oocyte and 2PN rates relative to the Small group(β>0, all P value < 0.05); and had less likely to develop into MI oocytes relative to the Small group(β<0, all P value < 0.05). The GLM indicated that AMH, along with Gn total dose, start dose, and Gn days, had significant impact on oocyte and embryo quality. For young patients, age was not a significant influencing factor, but for advanced-age patients, age influenced the outcomes. Our analysis suggests that for young patients (< 35 years), triggering when there is a high proportion of large or medium follicles results in better quality oocytes, while for older patients (≥ 35 years), it is better to trigger when the proportion of medium follicles is no less than that of small follicles. Further research is required to confirm these findings.

OXIDATIVE PROCESSES IN GRANULOSA CELLS OF COW OVARIAN FOLLICLES AT FOLLICULAR GROWTH

Article presents the research results on the respiratory activity and redox capacity of granulose layer cells of cow ovarian follicles under the physiological state of follicular growth. After the slaughter of the cows, the ovaries in physiological state of follicular growth were selected and the size of the follicles was evaluated. Cells of the granulosa layer were aspirated from small (&lt; 4 mm), medium (4–7 mm) and large (&gt; 7 mm) follicles and determined: intensity of oxygen absorption – polarographically, redox capacity – potentiometrically. K3[Fe(CN)6] was used as an extracellular electron acceptor. The share of oxygen consumed by aerobic glycolysis was determined by using sodium fluoride inhibitor, the NAD-dependent link of the mitochondrial electron transport chain - amytal, and the terminal (cytochrome oxidase) – sodium azide. The share of free-radical oxidation of unsaturated fatty acids in the realization of oxygen was detected by Na2EDTA.&#x0D; Granulosa cells from the ovary of follicular growth are characterized by respiratory activity and regenerative capacity, the intensity of which depends on the size of the follicles from which they are extracted. The highest values of the investigated indicators are characteristic of cells extracted from large follicles, lower – from small follicles, and the lowest values are from medium follicles. The high intensity of oxidative processes in granulosa from small follicles ensures the growth of oocytes, and from large follicles – maturation of the germ cell in the dominant follicle. At the same time, the increased intensity of oxygen consumption by the cells of small and dominant follicles is manifested against the background of limitations in electron acceptors in the extracellular environment.&#x0D; The use of inhibitors of ATP resynthesis of both glycolysis and individual links of the mitochondrial respiratory chain testify the activity of the indicated metabolic pathways in granulosa and the possibility of using alternative substrates to ensure the existence of cells. The main way of utilization of oxygen in the granulosa layer cells of small and large follicles is aerobic glycolysis, and from the medium there are alternative ways of supplying and using substrates. The largest flow of reducing equivalents into the extracellular space from the granulosa of a medium-sized follicle due to inhibition of glycolysis indicates that the source of the supply of reducing equivalents into the follicle cavity is glycolysis and this characterizes the dependence of the existence of cells on the indicated metabolic pathway.

Determination of the appropriate concentration of sodium alginate used for in vitro culture of cat preantral follicles in a serum-free medium containing FSH, EGF and IGF-I.

Culture of domestic cat preantral follicles can be a suitable technology to assist oocyte conservation strategies in the family Felidae. This research was aimed to comparatively analyse cat preantral follicular development of follicles directly seeded on growth surface or encapsulated in 0.5 or 1% of sodium alginate in a serum-free medium containing FSH, EGF and IGF-I. Preantral follicles were isolated from cat ovarian cortical tissue after ovariectomy. Alginate was dissolved at 0.5 or 1% in PBS. Follicles, 4 per well, with 0% (G-0%), 0.5% (G-0.5%) or 1% (G-1%) of sodium alginate were cultured in M199 with FSH (100 ng/mL), EGF (100 ng/mL) and IGF-I (100 ng/mL) for 7 days at 37°C, 5% CO2 and 99% humidity. Culture medium was replaced every 48h and samples were stored at -20°C until ELISA of steroid hormones. Morphometric evaluation of follicles was performed every 24h. G-0% follicles showed granulosa cell migration away from the oocyte and disrupted morphology, whereby they reached apparently larger diameters (203.70 ± 5.82 μm; p < .05) than G-0.5% and G-1% follicles (157.89 ± 8.47 μm and 95.23 ± 1.67 μm, respectively) which maintained three-dimensional organization, being larger in G-0.5% than in G-1% (p < .05). G-0.5% follicles attained the multi-layer preantral follicle stage on day 7 of culture, whereas G-1% follicles underwent progressive atresia. On day 6, steroid concentrations were higher (p < .05) in G-0% than in G-1%: 60 ± 19 vs 0.88 ± 0.32 pg/mL oestradiol; 2.6 ± 0.84 vs 0.04 ± 0.02 ng/mL progesterone; 1.3 ± 0.22 vs 0.61 ± 0.04 ng/mL testosterone and 1.6 ± 0.54 vs 0.22 ± 0.07 ng/mL androstenedione respectively. Steroid concentrations in G-0.5% were comprised between those of G-0% and G-1% (p > .05). In conclusion, two-layer cat preantral follicles encapsulated in 0.5% alginate cultured in medium containing FSH, EGF and IGF-I can develop up to the multi-layer preantral stage in 7 days of culture, whereas follicles directly seeded on growth surface or encapsulated in 1% alginate lost their three-dimensional organization, and experienced regression with compromised steroidogenesis, respectively.

The impact of FSH stimulation and age on the ovarian and uterine traits and histomorphometry of prepubertal gilts

This study investigated the effect of age and follicle stimulating hormone (FSH) treatment on the estradiol (E2) plasma concentration, ovarian follicle development, endometrial histomorphometry, and ultrasonographic parameters of the ovaries and uterus in prepubertal gilts. Thirty-five prepubertal gilts were grouped according to age (140 or 160 d), and within each age, gilts were allotted to receive 100 mg of FSH (treated; G140 + FSH [n = 10] and G160 + FSH [n = 7]) or saline solution (control; G140 + control [n = 10] and G160 + control [n = 8]). The total dose of FSH was divided into 6 similar doses administered every 8 h (days 0–2). Before and after FSH treatment, blood sample was collected, and transabdominal scanning of the ovaries and uterus was performed. Twenty-four hours after the last FSH injection, the gilts were slaughtered and their ovaries and uterus were processed for histological and histomorphometric analysis. The histomorphometric parameters of the uterus differed (P < 0.05) between prepubertal gilts at 160 d and 140 d of age. Moreover, changes (P < 0.05) in uterine and ovarian ultrasound images occurred between 140 and 160 d of age. Age and FSH treatment did not affect (P > 0.05) E2 plasma concentrations. Follicle stimulating hormone treatment did not affect (P > 0.05) the early stage of folliculogenesis in the prepubertal gilts; however, the number of early atretic follicles decreased (P < 0.05) after the FSH treatment. Follicle stimulating hormone administration increased (P < 0.05) the number of medium follicles and decreased (P < 0.05) the number of small follicles in 140 and 160 d old gilts. In the endometrium, luminal/glandular epithelium height and glandular diameter increased (P < 0.05) after FSH treatment. Thus, injections of 100 mg of FSH stimulate the endometrium epithelium and induce follicular growth to a medium follicle size without affecting the preantral stages in prepubertal gilts; also, the uterine macroscopic morphometry does not change from 140 to 160 d of age.

The effect of age and FSH stimulation on the ovarian follicular response, nuclear maturation, and gene expression of cumulus-oocyte complexes in prepubertal gilts

This study investigated the effects of age and FSH treatment on the ovarian response, follicular fluid (FF) biochemical composition, nuclear maturation, and molecular profile of cumulus-oocytes complexes (COCs) recovered from prepubertal gilts. Thirty-five prepubertal gilts were separated according to age [140 (n = 20) or 160 (n = 15) days], and within each age, the gilts were allotted to receive either 100 mg of FSH [treated; G140+FSH (n = 10) and G160+FSH (n = 7)] or saline solution [control; G140+control (n = 10) and G160+control (n = 8)]. Thus, four experimental groups were included in this study. In the FSH-treated gilts, the percentage of medium follicles increased (P < 0.0001) in the same proportion with which the percentage of small follicles decreased (P < 0.0001). In addition, the glucose concentration in the FF obtained from medium follicles increased (P < 0.05), while that of triglycerides decreased (P < 0.05) in the FSH-treated gilts. The FSH stimulation also improved (P < 0.05) the number of grade I COCs obtained from medium follicles and the meiotic maturation and BCB + rates. FSH treatment only upregulated (P < 0.05) HMGCR expression in immature COCs from prepubertal gilts. The metaphase II and BCB + rates, FF glucose and plasma IGF-1 levels were greater (P < 0.05) in prepubertal gilts at 160 than at 140 days of age. Age had no effect (P > 0.05) on the transcript abundance of the target genes in immature COCs. Hence, oocytes obtained from 140-day-old prepubertal gilts appeared less meiotically competent than those of 160-day-old prepubertal gilts. Our study suggests a possible strategy of using FSH treatment to improve oocyte quantity, quality, and nuclear maturation in 140 and 160-day-old prepubertal gilts.

SUPEROVULATORY RESPONSE IN DAIRY COWS AFTER APPLICATION OF ECG

PURPOSE: The aim of the present study was to determine the follicular dynamics and superovulatory response in dairy cows after the administration of eCG. METHODS: The study included 6 Holstein cows bearing CL in the ovaries treated with PGF2α for estrus induction. After estrus detection daily ultrasound scanning to monitor the dominant follicle of the first follicular wave was made. Superovulation treatment (2500 IU eCG and two doses of 500μg PGF2α analog on the 48th and 60th hour after the eCG injection) started at the emergence of the second follicular wave. The number and diameter of the follicles ≥ 5 mm, the number of corpora lutea and anovulatory follicles (AF) and the ovulation rate (OR) were estimated. RESULTS: At the start of the superovulation treatment (SO) the average number and diameter of the medium and large follicles were 2.7±1.2 and 0.67±0.13 cm and 1.6±0.5 and 1.45±0.28 cm, respectively. There were high positive correlations between the days of the treatment and number of large and medium follicles (r = 0.80, P = 0.018 and r = 0.75, P = 0.03). The average number of large follicles on day 5 was greater than those at the start of SO. On day 8 the OR was 55.9% and the difference between anovulatory follicles and corpora lutea was not significant. CONCLUSIONS: The used protocol provided acceptable OR, but the average number of AF (44.1%) remained high. Ultrasonography could be used to monitor the first dominant follicle and the emergence of a new follicular wave.

Expression and localization of apelin and apelin receptor (APJ) in buffalo ovarian follicles and corpus luteum and the in-vitro effect of apelin on steroidogenesis and survival of granulosa cells

Apelin is an adipose tissue-derived hormone with many physiological functions, including the regulation of female reproduction. It acts through an orphan G protein-coupled receptor APJ/APLNR. The present study aimed to investigate the expression of apelin and its receptor APJ in the ovarian follicles and corpus luteum (CL) and the role of apelin on steroidogenesis and cell survival. Ovarian follicles were classified into four groups based on size and estradiol (E2) level in the follicular fluid as follows: (i) F1 (4–6 mm; <0.5 ng/mL) (ii) F2 (7–9 mm; 0.5–5 ng/mL) (iii) F3 (10–13 mm; 5–40 ng/mL) and (iv) F4 (dominant/pre-ovulatory follicle) (>13 mm; >180 ng/mL). The corpora lutea (CL) were categorized into early (CL1), mid (CL2), late luteal (CL3), and regressing (CL4) CL stages. Expression of apelin increased with follicle size, with significantly greatest in the dominant or pre-ovulatory follicle (P < 0.05). Expression of APJ was greater in large and dominant follicles than in small and medium follicles (P < 0.05). In CL, the mRNA and protein abundance of apelin and apelin receptor was greater during mid (CL2) and late luteal (CL3) stages as compared to early (CL1) and regressing (CL4) stages (P < 0.05). Both the factors were localized in granulosa and theca cells of follicles and small and large luteal cells of CL. The pattern of the intensity of immunofluorescence was similar to mRNA and protein expression. Granulosa cells were cultured in vitro and treated at 1, 10, and 10 ng/mL apelin-13 either alone or in the presence of the follicle-stimulating hormone (FSH) (30 ng/mL) or insulin-like growth factor-I (IGF-I) (10 ng/mL) for 48 h. The luteal cells were treated with apelin-13 at 1, 10, and 100 ng/mL doses for 48 h. Apelin treatment at 10 and 100 ng/ml significantly (P < 0.05) increased E2 secretion, cytochrome P450 aromatase or CYP19A1 expression in GC. In luteal cells, apelin at 10 ng/mL and 100 ng/mL significantly (P < 0.05) increased progesterone (P4) secretion and HSD3B1 expression. In GCs, apelin, either alone or in combination, increased PCNA expression and inhibited CASPASE3 expression suggesting its role in cell survival. In conclusion, this study provides novel evidence for the presence of apelin and receptor APJ in ovarian follicles and corpora lutea and the stimulatory effect on E2 and P4 production and promotes GC survival in buffalo, suggesting the role of apelin in follicular and luteal functions in buffalo.

HISTOTOPOGRAPHIC AND STRUCTURAL FEATURES OF CANINE THYROID GLAND FOLLICLES UNDER SINGLE DYNAMIC MOTOR LOADS

The formation of urgent and long-term adaptation of the organism under various modes of motor loads is provided by a change in the activity of the thyroid gland, the functional unit of which is the thyroid follicle. An accurate assessment of the morphological parameters of the follicles of different histotopographic zones of the thyroid gland provides the researcher with objective information about the state of the organ. The purpose of the study was to evaluate the effect of single training, starting and limiting motor loads (treadmill running) on ​​the structural and histotopographic features of the thyroid gland follicles in dogs. On transverse sections of the right lobe of the thyroid gland of dogs, stained with hematoxylin-eosin, the area of ​​the follicles were measured. The measurements were carried out in various histotopographic zones of the gland (central, intermediate, peripheral), distinguished relative to its geometric center. Animals were studied in the control group (16 male dogs) and in the experiment (31 animals), in which, as a result of single motor loads, a different response was formed, manifested in structural and functional changes in the thyroid gland. The studies showed that muscle activity leads to the activation of the functional units of the organ - follicles. The transformation of the thyroid gland as a result of various durations of motor loads is manifested in the obligate involvement of the peripheral zone of the gland, a significant decrease in the area of ​​follicles, an increase in the number of functionally more active follicles of a small area, due to a decrease in the number of predominantly large and medium follicles, and changes in the colloid-epithelial areas ratio. Depending on the parameters of single dynamic motor loads, two ways of primary response of the gland were revealed: either due to a change in the number of follicles of different areas (large, medium, small), or due to a change in their ratio in the histotopographic zones of the organ (central, intermediate, peripheral). Modeling of various parameters of motor loads can be used to analyse its of the influence the structural reorganization of the thyroid gland in the experiment. The constant structural variability of the peripheral zone found in the present study may be associated with special conditions of blood supply from the gland capsule.

Hormone regulation of thrombospondin-1 mRNA in porcine granulosa cells in vitro

Thrombospondin-1 (THBS1) is involved in the process of angiogenesis and is down-regulated by insulin-like growth factor 1 (IGF1) in porcine granulosa cells (GC), but what other hormones regulate GC THBS1 and its role in follicular growth is unclear. Thus, six experiments were conducted to determine the influence of other hormones on THBS1 gene expression in porcine GC, and to determine if THBS1 mRNA changes during follicular development. For Exp. 1–5, small (1–5 mm) follicles from ovaries of abattoir gilts were aspirated, GC collected and treated with FSH, IGF1, fibroblast growth factor 9 (FGF9), Sonic hedgehog (SHH), estradiol, cortisol, and/or prostaglandin E2 (PGE2). FSH, IGF1 and FGF9 each decreased (P < 0.05) THBS1 mRNA abundance. Alone, PGE2 increased (P < 0.05) THBS1 mRNA abundance. PGE2 significantly attenuated the FSH-induced inhibition of THBS1 mRNA expression. Estradiol, cortisol, and SHH had no effect on THBS1 mRNA abundance. In Exp. 6, small (1–3 mm), medium (4–6 mm) and large (7–14 mm) follicles were aspirated to measure abundance of THBS1 mRNA in GC which did not differ (P > 0.10) between small and medium-sized follicles but was threefold greater (P < 0.05) in large compared to small or medium follicles. We hypothesize that the inhibitory effects of FSH, IGF1 and FGF9 on the antiangiogenic gene THBS1 could contribute to promoting angiogenesis in the developing follicle, while stimulation of THBS1 mRNA by PGE2 may help reduce angiogenesis during the preovulatory period when PGE2 and THBS1 mRNA are at their greatest levels.

Nanodelivery System for Ovsynch Protocol Improves Ovarian Response, Ovarian Blood Flow Doppler Velocities, and Hormonal Profile of Goats.

Simple SummaryDrug delivery systems depending on nanotechnology have been recently created to improve biological activity of drugs including hormones. Nanoformulated drugs have different pharmacokinetic properties in biological systems compared to their conventional forms due to the acquired new physicochemical properties such as lesser size, high size-to-weight ratio, and different surface charges and shapes. These properties enable them to efficiently deliver into target sites coping with existing biological barriers. Ovsynch protocol, GPG, is one of the most important estrous synchronization protocols due to the possibility of applying timed insemination with this protocol. This protocol is commonly used in many farm animals, as it aids in herd management and eliminates the need for the detection of estrus. However, there are still some shortcomings that restrict the outcomes of this protocol, such as scattering ovulation time, short luteal phase and inadequate luteal function, and low conception rates. Therefore, this study aims to evaluate ovarian response, blood flow of the ovarian and luteal arteries, and hormonal profile of goats receiving either a standard Ovsynch protocol or an Ovsynch protocol delivered via a nanodelivery system with different dosages of hormones. The present study is the first one that poses the idea that the nanodelivery system for the Ovsynch protocol enables lower hormone dose administration and improves the results of the protocol by inducing tighter synchrony of ovulation and better luteal function of synchronized goats.Fifteen cyclic, multiparous goats were equally stratified and received the common Ovsynch protocol (GPG: intramuscular, IM, injection of 50 mg gonadorelin, followed by an IM injection of 125 µg cloprostenol 7 days later, and a further IM injection of 50 mg gonadorelin 2 days later) or the Ovsynch protocol using nanofabricated hormones with the same dosages (NGPG) or half dosages (HNGPG) of each hormone. The ovarian structures and ovarian and luteal artery hemodynamic indices after each injection of the Ovsynch protocol using B-mode, color, and spectral Doppler scanning were monitored. Levels of blood serum progesterone (P4), estradiol (E2), and nitric oxide (NO) were determined. After the first gonadotrophin-releasing hormone (GnRH) injection, the number of large follicles decreased (p = 0.02) in NGPG and HNGPG, compared with GPG. HNGPG resulted in larger corpus luteum (CL) diameters (p = 0.001), and improved ovarian and luteal blood flow, compared with GPG and NGPG. Both NGPG and HNGPG significantly increased E2 and NO levels compared with GPG. HNGPG increased (p < 0.001) P4 levels compared with GPG, whereas NGPG resulted in an intermediate value. After prostaglandin F2α (PGF2α) injection, HNGPG had the largest diameter of CLs (p = 0.001) and significantly improved ovarian blood flow compared with GPG and NGPG. Both NGPG and HNGPG increased (p = 0.007) NO levels, compared with GPG. E2 level was increased (p = 0.028) in HNGPG, compared with GPG, whereas NGPG resulted in an intermediate value. During the follicular phase, HNGPG increased (p = 0.043) the number of medium follicles, shortened (p = 0.04) the interval to ovulation, and increased (p < 0.001) ovarian artery blood flow and levels (p < 0.001) of blood serum P4, E2, and NO, compared with GPG and NGPG. During the luteal phase, the numbers of CLs were similar among different experimental groups, whereas the diameter of CLs, luteal blood flow, and levels of blood serum P4 and NO increased (p < 0.001) in HNGPG, compared with GPG and NGPG. Conclusively, the nanodelivery system for the Ovsynch protocol could be recommended as a new strategy for improving estrous synchronization outcomes of goats while enabling lower hormone dose administration.