Follicular Population Research Articles

Determinants of the follicular population and oocyte quality of Cameroonian native ewes

Determinants of the follicular population and oocyte quality of Cameroonian native ewes

224 Superstimulation effect on invitro embryo production and relationship with anti-Müllerian hormone in buffaloes (Bubalus bubalis) aspirated 7 or 14 days after follicular ablation

The success of buffalo invitro embryo production (IVEP) depends, in part, on the number and competence of oocytes. Superstimulation with FSH has been linked to oocyte quality and embryo yield, and anti-Müllerian hormone (AMH) has been proposed as an indicator of ovarian reserve. The objective was to evaluate FSH stimulation in one ovum pick-up (OPU) cycle conducted 7 or 14 days after follicular ablation; we additionally analysed residual FSH treatment effect carried over to a subsequent aspiration cycle. Finally, we evaluated the relationship between plasma AMH and FSH response. Nine multiparous, cycling Murrah donors (603±7.0kg, body condition score=3.5) were subjected to complete follicular ablation followed by OPU 1 week later (T0 Control), and plasma AMH was determined at this point. They were then randomly allocated to FSH stimulation (3 days b.i.d. in descending doses, total 300mg of pFSH, Vetpharm, with the last injection 48h before OPU) with aspiration conducted 7 (T7 FSH, n=5) or 14 days (T14 FSH, n=4) post-follicular ablation. The residual effect (T Residual, n=5) was evaluated in one subsequent, nonstimulated OPU cycle conducted 1 week later (9 days after last FSH administration). For plasma AMH, concentration was measured using ELISA kit (cat. no. AL114, AnshLabs; sensitivity 1.0 pgmL−1, interassay variation <5%, additional validation conducted with serial dilutions of buffalo plasma, r=0.98 against standard curve). Continuous data were analysed with ANOVA and Tukey's post hoc comparisons; categorical data were analysed using Chi-squared test. Pearson coefficient was used to evaluate correlation between AMH, follicular reserve, and invitro performance. Superovulatory treatment resulted in a higher number of follicles and good quality oocytes (grades 1 and 2) per donor, irrespective of OPU interval (T7 FSH: 3.0±2.55 and T14 FSH: 1.5±0.5 vs. T0 Control: 0.45±0.38 and T Residual: 0.4±0.4; P<0.05). There were more cleaved embryos (T7 FSH: 26%, T14 FSH; 30% vs. T0 Control: 7% and T Residual: 20%) and blastocysts (T7 FSH: 21%, T14 FSH; 30% vs. T0 Control: 5% and T Residual: 0; P<0.05) from FSH-treated donors. There was a strong correlation (r=0.9439) between follicle count and plasma AMH, and weaker relationships for oocytes recovered (r=0.3975), cleaved embryos (r=0.2317), or blastocysts (r=0.0106). Results indicate that FSH treatment with a 48-h coasting period results in significant improvements in follicular population, oocyte quality, and embryonic development; these effects are observed independently of the interval between initial follicular ablation and OPU. No residual effects of FSH treatment were observed, indicating that the beneficial effects of superstimulation are not carried over to the following OPU cycle. In conclusion, a strong correlation coefficient was established between initial plasma AMH level and follicular response to FSH treatment, indicating that it may be used as a selection criterion for animals likely to exhibit a good response to superstimulatory treatment.

128 Study of preantral ovarian follicular population in fetal alpaca (Vicugna pacos) ovaries

In mammals, folliculogenesis begins at the fetal stage and is a complex, dynamic process that involves follicular quiescence, activation, growth, follicular migration, and cell interactions. At birth, the preantral ovarian follicular population, drastically reduced, constitutes >90% of all ovarian follicles, representing the ovarian reserve that will be used throughout the reproductive life. In alpacas, changes in follicular wave growth patterns and ovulation are different from other species; thus, it is important to know the ovarian reserve at fetal stage, as a starting point for future studies. Therefore, the aim of the present study was to establish the morphological characterisation and estimate the population of alpaca preantral follicles in the fetal stage. Ovaries from alpacas (n=5) in the fetal stage (fetus during the last third of gestation) were collected at a local slaughterhouse. Whole ovaries were individually fixed in 4% paraformaldehyde phosphate-buffered saline overnight at room temperature for routine histology. Ovaries were dehydrated in alcohol, cleared with xylene, and embedded in paraffin, and all tissue was serially sectioned at 7μm with a rotating microtome (Leica). The histological sections were mounted and stained with periodic acid-Schiff and hematoxylin. Preantral follicles were classified according to their developmental stage: primordial (one layer of flattened granulosa cells surrounding the oocyte), primary follicle transition (flattened cuboidal granulosa cells surrounding the oocyte), primary (single layer of cuboidal granulosa cells around the oocyte), or secondary (oocyte surrounded by more than one complete layer of cuboidal granulosa cells). We estimated the number of preantral follicles by counting all follicles in each histological section. Only follicles in which the oocyte nucleus was visible were counted. In addition, for each follicle category (n=40 per group), oocyte and follicle diameters were measured using an ocular micrometer. The variable means were compared using unpaired Student's t-test analysis, with significance set at P ≤ 0.05. Estimation of preantral follicular population (mean±standard deviation) was 80 516±14 575 in the ovaries of alpaca fetuses. Most of the follicles found belong to the primordial (49.2%) or primary follicle transition (39.2%) categories, followed by primary (10.8%) and secondary (0.8%) stages. Follicle and oocyte diameters of primordial (33.3±7.2; 21.5±4.6μm) and primary follicle transition stages (36.7±3.0; 23.4±2.6μm) were significantly (P<0.05) smaller than those of primary-stage follicles (77.9±15.8; 50.02±11.1μm). Finally, preantral follicles classified with normal morphological integrity appearance for each developmental stage were 98.2% (primordial), 96.7% (primary follicle transition), 91.5% (primary), and 88.1% (secondary), respectively. In conclusion, this study shows for the first time an estimation of the population of preantral follicles in alpaca fetal ovaries and establishes follicle and oocyte diameters and their normal morphological integrity.

Efficacy of repeated ovum pick-up in Podolic cattle for preservation strategies: a pilot study

The study evaluated the effects of eCG treatment prior to ovum pick up (OPU) on follicular population, oocyte and embryo yields in summer and autumn in Podolic cattle. The effects of repeated OPU on cattle wellbeing was also documented. Twenty-six animals were used, and split into two groups, treatment (OPU; n = 18) and control (CG; n = 8). The OPU cattle were subsequently split into two subgroups (n = 9) and underwent repeated OPU, without and with eCG priming, for a total of 8 sessions (4 sessions/season). Follicular population, oocyte and embryo yields were recorded in those sub-groups. CG was handled in the same manner of OPU, except for epidural anaesthesia and follicular aspiration. Biochemical profile, serum protein electrophoresis and haptoglobin levels were analysed in OPU and CG. Hormonal priming increased the number of medium follicles (1.7 ± 0.2 vs 0.9 ± 0.2, p < .05), while it decreased the recovery rate and number of cumulus-enclosed oocytes (COCs) (recovery rate: 38.3 ± 3.5 vs 60.5 ± 4.0%; COCs: 2.3 ± 0.3 vs 3.4 ± 0.4, respectively; p < .01). However, priming increased cleavage (72.9 ± 5.7 vs 49.4 ± 5.4; p < .05) and blastocyst (41.1 ± 5.7 vs 23.0 ± 4.2; p = .054) rates. With regard to season’s effect, a higher number of COCs was recorded in autumn than in summer (3.1 ± 0.4 vs 2.6 ± 0.3; p < .05) without affecting though the number of embryos produced (0.9 on average). Since haematological parameters did not vary between OPU and CG, our preliminary data suggest that repeated OPU may be used as a conservation strategy in Podolic cattle without affecting wellbeing. Highlights Podolic cattle is an endangered Italian breed, reared in semi-extensive/extensive systems Ovum pick-up (OPU) can be carried out in this breed without impairing animal health and welfare The eCG treatment before OPU (priming) did not improve the number of embryos produced per donor.

Common variable immunodeficiency–associated endotoxemia promotes early commitment to the T follicular lineage

Although chiefly a B-lymphocyte disorder, several research groups have identified common variable immunodeficiency (CVID) subjects with numeric and/or functional TH cell alterations. The causes, interrelationships, and consequences of CVID-associated CD4+ T-cell derangements to hypogammaglobulinemia, autoantibody production, or both remain unclear. We sought to determine how circulating CD4+ Tcells are altered in CVID subjects with autoimmune cytopenias (AICs; CVID+AIC) and the causes of these derangements. Using hypothesis-generating, high-dimensional single-cell analyses, we created comprehensive phenotypic maps of circulating CD4+ T cells. Differences between subject groups were confirmed in a large and genetically diverse cohort of CVID subjects (n=69) by using flow cytometry, transcriptional profiling, multiplex cytokine/chemokine detection, and a suite of invitro functional assays measuring naive T-cell differentiation, B-cell/T-cell cocultures, and regulatory T-cell suppression. Although CD4+ TH cell profiles from healthy donors and CVID subjects without AICs were virtually indistinguishable, T cells from CVID+AIC subjects exhibited follicular features as early as thymic egress. Follicular skewing correlated with IgA deficiency-associated endotoxemia and endotoxin-induced expression of activin Aand inducible T-cell costimulator ligand. The resulting enlarged circulating follicular helper T-cell population from CVID+AIC subjects provided efficient help to receptive healthy donor B cells but not unresponsive CVID B cells. Despite this, circulating follicular helper T cells from CVID+AIC subjects exhibited aberrant transcriptional profiles and altered chemokine/cytokine receptor expression patterns that interfered with regulatory T-cell suppression assays and were associated with autoantibody production. Endotoxemia is associated with early commitment to the follicular T-cell lineage in IgA-deficient CVID subjects, particularly those with AICs.

In vitro survival of follicles in prepubertal ewe ovarian cortex cryopreserved by slow freezing or non-equilibrium vitrification.

Vitrification is a well-accepted fertility preservation procedure for cryopreservation of oocytes and embryos but little is known regarding ovarian tissue, for which slow freezing is the current convention. The aim of the present study was to assess the efficiency of non-equilibrium vitrification compared to conventional slow freezing for ovarian cortex cryopreservation. Using prepubertal sheep ovaries, the capacity of the tissue to sustain folliculogenesis following cryopreservation and in vitro culture was evaluated. Ovarian cortex fragments were cultured in wells for 9days, immediately or after cryopreservation by conventional slow freezing or non-equilibrium vitrification in straws. During culture, follicular populations within cortex were evaluated by histology and immunohistochemistry for PCNA and TUNEL. Steroidogenic activity of the tissue was monitored by assay for progesterone and estradiol in spent media. No significant differences in follicle morphology, PCNA, or TUNEL labeling were observed between cryopreservation methods at the initiation of culture. Similar decreases in the proportion of primordial follicle population, and increases in the proportion of growing follicles, were observed following culture of fresh or cryopreserved ovarian tissue regardless of cryopreservation method. At the end of culture, PCNA and TUNEL-positive follicles were not statistically altered by slow freezing or vitrification in comparison to fresh cultured fragments. Overall, for both cryopreservation methods, the cryopreserved tissue showed equal capacity to fresh tissue for supporting basal folliculogenesis in vitro. Taken together, these data confirm that both non-equilibrium vitrification and slow-freezing methods are both efficient for the cryopreservation of sheep ovarian cortex fragments.

Single-cell reconstruction of follicular remodeling in the human adult ovary

The ovary is perhaps the most dynamic organ in the human body, only rivaled by the uterus. The molecular mechanisms that regulate follicular growth and regression, ensuring ovarian tissue homeostasis, remain elusive. We have performed single-cell RNA-sequencing using human adult ovaries to provide a map of the molecular signature of growing and regressing follicular populations. We have identified different types of granulosa and theca cells and detected local production of components of the complement system by (atretic) theca cells and stromal cells. We also have detected a mixture of adaptive and innate immune cells, as well as several types of endothelial and smooth muscle cells to aid the remodeling process. Our results highlight the relevance of mapping whole adult organs at the single-cell level and reflect ongoing efforts to map the human body. The association between complement system and follicular remodeling may provide key insights in reproductive biology and (in)fertility.

Superstimulation prior to the ovum pick-up improves the in vitro embryo production in nulliparous, primiparous and multiparous buffalo (Bubalus bubalis) donors

The aim of this study was to evaluate the ovarian follicular population, the oocyte yield and the in vitro embryo production (IVEP) of nulliparous (NU), primiparous (PR) and multiparous (MU) buffalo donors submitted to the superstimulation with FSH prior to the ovum pick-up (OPU). A total of 54 buffalo donors (18 NU, 15 PR and 21MU) received an intravaginal progesterone device (1.0 g) plus estradiol benzoate [2.0 mg, intramuscular (im)] at random stage of the estrous cycle (Day 0) during the breeding season (autumn and winter). Buffaloes from different categories were then randomly allocated to one of two groups (Control or FSH), in a cross-over experimental design. Buffalo donors in the Control group received no further treatment, whereas buffalo donors in the FSH group received a total dosage of 200 mg im of FSH on Days 4 and 5, in four decreasing doses 12 h apart (57, 57, 43 and 43 mg). On Day 7, the progesterone device was removed and the OPU procedure was performed in both groups. The same semen was used across all replicates and donor category. Data were analyzed by the GLIMMIX procedure of SAS 9.4®. There was no interaction between FSH treatment and animal category for all analyzed variables. Furthermore, no differences between animal category (P = 0.73) and FSH treatment (P = 0.53) were observed regarding the total follicles aspirated. However, the FSH treatment increased (P < 0.001) the proportion of large (>10 mm; FSH = 16.2% and Control = 2.0%) and medium-sized follicles (6–10 mm; FSH = 36.3% and Control = 6.1%) available for the OPU procedure. The total of recovered oocytes was greater in NU than in MU, and PR were similar to NU and MU (P = 0.05). No effect of FSH treatment was observed (P = 0.85) for this variable. Buffalo donors treated with FSH had a greater viable oocytes rate (P = 0.03), blastocyst rate (P = 0.03) and embryo yield per OPU-IVEP session (P = 0.07), however, no category effects were observed for these variables. These results provided evidence that superstimulation with FSH increased the proportion of large and medium-sized follicles available for the OPU procedure. Consequently, the FSH treatment enhanced the proportion of viable oocytes for culture and resulted in greater blastocyst rates and embryo yield per OPU-IVEP session in all buffalo donors categories.

Endocrine Profile and Reproductive Performance in Heifers Induced to Lactation

Background: Low reproductive efficiency has been one of the main factors that lead to dairy herd culling in the reproductive age. In multiparous animals, such inefficiency and culling can occur because of various factors and may be associated with occasional endocrine failures. To avoid revenue losses that incur due to lack of lactation, lactation is artificially induced in the animals that are not pregnant, using a defined protocol. The aim of this study was, therefore, to evaluate and compare the reproductive performance of heifers submitted to the induction protocol with that of pregnant heifers during the transition period.Materials, Methods &amp; Results: Sixty Holstein heifers, 32 ± 0.6 months of age, were divided into two groups: Control Group (Control, n = 30) comprising pregnant heifers that were accompanied since 21 days before the expected calving date until 224 days in milk (DIM) and an Induction Group (Induction, n = 30) comprising non-pregnant heifers submitted to a lactation induction protocol, accompanied from the beginning of the protocol until 224 DIM. For evaluation of the endocrine profile (progesterone and estradiol concentrations) of these animals, blood samples were collected at two periods: the pre-lactation (weeks -3, -2 and -1) and post-lactation (weeks 1 and 4). Heifers from both groups were submitted to weekly reproductive evaluations, from the beginning of lactation until 35 DIM. Uterine examinations were performed using ultrasonography and vaginoscopy to evaluate uterine content, cervical opening, and mucosal aspect. Females in good reproductive health were subjected to a hormonal protocol for timed artificial insemination (TAI). The pregnancy rate of heifers that could reproduce (Control, n = 13, and Induction, n = 20) were evaluated and inseminated until 49 DIM. Progesterone levels were similar (P &gt; 0.05) in the two groups at both pre- and post-start of lactation. Estradiol concentrations were different (P &lt; 0.01) among groups only in the pre-lactation period. Higher levels of progesterone (P = 0.06) were observed in the induced heifers (Induction Group = 1.07 ± 0.23 ng/mL and Control Group = 0.38 ± 0.28 ng/mL). Therefore, in the fourth week, induced heifers exhibited higher luteinic activity, the progesterone concentration was above 1 ng/mL in 42% of the animals, while just 12.5% of the Control heifers had it. The overall pregnancy rate was 47.75%. The pregnancy rate recorded for induced heifers and Control heifers was 40% and 55.55%, respectively.Discussion: Progesterone and estradiol evaluations were performed in the first week after the beginning of the lactation to evaluate the metabolism and physiological concentration of the hormones used during the protocol. The measurements were again performed in the fourth week to evaluate the return to cyclicity of the heifers in both groups. The high serum concentrations of estradiol attained during the induction, in the pre-lactation period, may have possible interfered with the uterine environment and follicular population of these animals. Furthermore, it is possible that the hormonal combination used has a central effect on the synthesis and release of gonadotrophins and/or an effect on the ovarian follicular population. Based on the hormonal profile evaluated, particularly the endocrine profile, and the reproductive performance of heifers, the results suggest that the protocol for induced lactation has a positive effect on fertility.

Effects of Breed, Age, Body Condition Score, and Nutritional Status on Follicular Population, Oocyte Yield, and Quality in Three Cameroonian Zebus CattleBos indicus

This study was aimed at investigating the effects of breed, age, body condition score, and nutritional status on follicular dynamics, quality, and oocyte yield in Cameroonian zebus cattleBos indicus(Gudali and Red and White Fulani). A total of 95 cows were investigated for breed, age, body condition score, and nutritional status. Blood samples and ovaries of these cows were collected during slaughtering. Follicles on each ovary were counted and measured using electronic digital calipers and then classified into three categories: small (&lt;3 mm), medium (3 – 8 mm), and large (&gt; 8 mm) diameter. The oocytes were retrieved by slicing each ovary into a Petri dish containing collected medium. The oocytes recovered were examined under a stereoscope (x 10) and classified into four groups based on the morphology of cumulus oophorus cells and cytoplasmic changes. From these ovaries, 3888 follicles were counted with an average population of 27.81 ± 6.88 follicles per cow. The average numbers of small, medium, and large follicles per cow were 17.26 ± 1.36 (62.06%), 9.64 ± 1.00 (34.66%), and 0.52 ± 0.32 (1.86%), respectively. A total of 1822 oocytes were harvested with an average of 19.11 ± 0.89 oocytes per cow. Oocytes graded I, II, III, and IV were 7.03 ± 2.30 (36.65%), 4.53± 2.01 (23.61%), 3.19 ± 0.85 (16.63%), and 4.43 ±1.62 (23.09%), respectively. This study also showed that ovarian weight, number of follicles, and oocytes’ yield of right ovary were higher than left ones (P &lt; 0.05). Younger and normal cows (BCS = 3) presented higher (P &lt; 0.05) number of follicles and oocytes. Urea and cholesterol levels were higher in Gudali and White Fulani cows (P &lt; 0.05). This study indicated that ovaries of zebus harvested in slaughterhouse are an important source of follicular population, oocytes yield, and quality. Right ovaries are heavy and offer more numbers of follicles and oocytes than the left ones. BCS is the best indicator of nutritional status of cows. Important follicle populations are obtained from cows aging 6-9 years with BCS = 3.

Hypoxia-inducible factors in CD4+ T cells promote metabolism, switch cytokine secretion, and T cell help in humoral immunity

T cell help in humoral immunity includes interactions of B cells with activated extrafollicular CD4+ and follicular T helper (Tfh) cells. Each can promote antibody responses but Tfh cells play critical roles during germinal center (GC) reactions. After restimulation of their antigen receptor (TCR) by B cells, helper T cells act on B cells via CD40 ligand and secreted cytokines that guide Ig class switching. Hypoxia is a normal feature of GC, raising questions about molecular mechanisms governing the relationship between hypoxia response mechanisms and T cell help to antibody responses. Hypoxia-inducible factors (HIF) are prominent among mechanisms that mediate cellular responses to limited oxygen but also are induced by lymphocyte activation. We now show that loss of HIF-1α or of both HIF-1α and HIF-2α in CD4+ T cells compromised essential functions in help during antibody responses. HIF-1α depletion from CD4+ T cells reduced frequencies of antigen-specific GC B cells, Tfh cells, and overall antigen-specific Ab after immunization with sheep red blood cells. Compound deficiency of HIF-1α and HIF-2α led to humoral defects after hapten-carrier immunization. Further, HIF promoted CD40L expression while restraining the FoxP3-positive CD4+ cells in the CXCR5+ follicular regulatory population. Glycolysis increases T helper cytokine expression, and HIF promoted glycolysis in T helper cells via TCR or cytokine stimulation, as well as their production of cytokines that direct antibody class switching. Indeed, IFN-γ elaboration by HIF-deficient in vivo-generated Tfh cells was impaired. Collectively, the results indicate that HIF transcription factors are vital components of the mechanisms of help during humoral responses.

Advances and Challenges of Using Ovarian Preantral Follicles to Develop Biobanks of Wild Mammals.

The extinction rate of mammalian species has been accelerated in recent decades. It therefore is important to preserve and store genetic materials in cryobanks for research purposes and subsequent production of offspring through assisted reproductive techniques. Along with the systematic collection and storage of germplasm, research efforts focusing on in vitro culture to produce mature gametes are critical. Specifically, obtaining mature oocytes from in vitro culture of preantral follicles is a great option to enhance female fertility preservation since these early follicles represent 90%-95% of the follicular population on ovarian cortex biopsy. This review presents current advances and discusses limitations and prospects about isolation, cryopreservation/banking, and in vitro culture of preantral follicles from wild species.

EVALUATION OF CORPORA LUTEA EFFECT ON OVARIAN MORPHOMETRY, FOLLICULAR POPULATION AND BIOCHEMICAL PROFILE IN FOLLICULAR FLUID AND BLOOD OF SLAUGHTERED COWS

Cows are occupied huge economic importance worldwide. Impaired fertility in cows due to absence of corpus luteum (CL) faces huge challenge. The aim of this study was to determine the effect of CL on ovarian biometry, follicular population, hormonal and metabolic content of serum and follicular fluid (FF) of cows. Blood samples and 48ovarieswerecollected from cows slaughtered at winter season and classified according to the presence or absence of CL into two groups, ovaries with and without CL. The diameter of antral follicles was taken and classified into three subgroups, small, medium and large follicles. FF aspirated from each follicle group. 17β-Estradiol (E2), progesteron (P4), glucose, total proteins, total cholesterol (TC) and nitric oxide (NO) were estimated. Result showed that the ovarian biometry was higher in right than left ovaries without significant difference. However, the total follicular populations and ovarian dimensions and their weights were significantly higher in ovaries with CL. The average number of small and medium follicles was also significantly increased in the ovaries with CL. However, the number of LF was higher in cows without CL compared to cows with CL. Glucose, Total cholesterol (TC), proteins, E2 and P4 concentrations were higher in serum than FF. Serum E2 concentration was higher in LF and significantly reduced in ovaries had CL than without CL. On the other hand, P4 concentration in FF was lower in LF and significantly increased in ovaries with CL. Ovaries had CL showed elevation of FF glucose level; however, TC, protein and NO concentrations were lower than ovaries without CL. Hence, we concluded that CL presence effects on both morphometric and metabolic conditions of cows, ovaries.

Evaluation de la population folliculaire et de la qualité des ovocytes des vaches zébus (&lt;em&gt;Bos indicus&lt;/em&gt;) en anœstrus anovulatoire

L’étude, menée à l’abattoir municipal de Ngaoundéré, région de l’Adamaoua au Cameroun, visait à évaluer la population folliculaire et la qualité des ovocytes des vaches zébus locales en anœstrus anovulatoire. Sur 496 voies génitales examinées, 145 (29,2 %) présentaient un anœstrus anovulatoire. Le nombre moyen de follicules était de 37,5 ± 25,2 par vache. Le nombre de petits, moyens et grands follicules par vache était respectivement de 21,7 ± 18,7, 14,9 ± 12,1 et 0,9 ± 1,1. Les populations folliculaires des vaches cycliques ou en anœstrus anovulatoire étaient respectivement de 29,1 ± 18,1 et 41,9 ± 27,3 (p &lt; 0,05). Le nombre d’ovocytes par vache était respectivement de 15,5 ± 4,5 et 15,6 ± 3,8 (p &gt; 0,05) chez les vaches cycliques et en anœstrus anovulatoire. Le nombre d’ovocytes de grades I, II, III et IV était respectivement de 4,5 ± 4,1 (28,5 %), 4,1 ± 3,7 (26,2 %), 2,3 ± 2,4 (14,8 %) et 4,8 ± 5,6 (30,5 %). Cette étude a montré que l’anœstrus anovulatoire avait tendance à augmenter la population folliculaire mais n’avait aucun effet sur le nombre d’ovocytes récoltés. Le nombre d’ovocytes de grades I et II utilisables pour la maturation in vitro était significativement plus faible chez les vaches en anœstrus anovulatoire que chez les vaches cycliques (p &lt; 0,05).

Effect of recombinant bovine somatotropin (rbST) treatment on follicular population and development in non-lactating dairy cows.

The aim of this study was to evaluate the long-term effects of recombinant bovine somatotropin (rbST) on follicle population and ovulatory follicle development in non-lactating dairy cows. Twenty-one Jersey cows were allocated in rbST (n=11) or control (n=10) groups. On day -60, cows in rbST group received 500 mg of somatotropin (s.c. Lactotropin®, Elanco). On day 0, control and rbST cows received an intravaginal progesterone-releasing device (1.9 g, CIDR®, Zoetis) and GnRH (100 mg, IM, Factrel®, Zoetis). On day 8, cows received PGF2α (25 mg, IM, Lutalyse®, Zoetis) and the CIDR® was removed. Twelve hours after device removal (D8), serum, follicular fluid and granulosa cells samples were collected. Serum and follicular concentration of estradiol (E2) and progesterone (P4) were analyzed. Total RNA was extracted from granulosa cells to measure gene expression of LHCGR, STAR, HSD-3B1, CYP11A1, CYP19A1, CYP17A1, IGFR and PAPPA by real-time PCR. Ultrasonography was performed on days -60, -53, -46, -14, -7, 0 and 8 for antral follicle count. Results were analyzed by repeated measures ANOVA and t-test. There was no effect of rbST treatment on the number of follicles during the 60 days period, as well as no effect on serum and follicular fluid E2 and follicular fluid P4 at the moment of follicle aspiration. There was a reduction in PAPPA (P = 0.006), CYP11A1 (P = 0.04) and CYP19A1 (P = 0.002) mRNA levels in granulosa cells of the pre-ovulatory follicle of rbST treated cows. In conclusion, a single dose of rbST did not have long-term effects on antral follicle population, serum and follicular E2/P4 concentrations in non-lactating dairy cows. Despite that, rbST injection decreased granulosa cell expression of genes related to steroidogenesis in the pre-ovulatory follicle.

Cell-free therapy with the secretome of adipose tissue-derived stem cells in rats\u2019 frozen-thawed ovarian grafts

The use of secretome may be a new strand of cell therapy, which is equal to or even superior to the injection of live cells, called cell-free therapy. In ovarian transplantation, this approach may be a therapeutic possibility for the ovarian graft in hypoxia. We designed the present study to evaluate whether the cell-free therapy with the secretome of adipose tissue-derived stem cells (ASCs) in rat frozen-thawed ovarian grafts could protect a graft against ischemic injury. A single dose of rat ASCs secretome or vehicle was injected into the bilateral frozen-thawed ovaries of 18 adult female rats immediately after an autologous transplant. Nine animals were used to control the cryopreservation protocol and were evaluated before and after the cryopreservation process. Daily vaginal smears were performed for estrous cycle evaluation until euthanasia on postoperative day 30. Follicle viability by trypan blue, graft morphology by HE, and apoptosis by TUNEL and cleaved-caspase-3 were assessed. No differences were found with respect to estrous cycle resumption and follicle viability (p > 0.05). However, compared with the vehicle-treated grafts, the morphology of the secretome-treated grafts was impaired, showing reduced follicular population and increased apoptosis (p < 0.05). ASC secretome impaired the rat frozen-thawed ovarian graft from ischemic injury. However, more studies are needed to evaluate the factors involved and the possibility of applying the secretome in scaffolds to optimize its use.

Oocyte developmental competence is improved by relatively greater circulating progesterone concentrations during preovulatory follicular growth

This study evaluated the effect of progesterone priming during follicular growth on oocyte competence to undergo oocyte cleavage and embryo development in sheep. Two experiments were performed on a total of 195 females that either received or did not receive a progesterone treatment (CIDR-type device) during the first follicular wave, beginning soon after ovulation (i.e., Day 0 of the experiment). On Day 3, the follicular population and oocyte quality (Experiment 1 and 2) and the competence of oocytes for cleavage and embryo development (Experiment 2) were evaluated after laparoscopic ovum pickup (LOPU) and in vitro fertilization. In Experiment 1, in a 2 × 2 factorial study the progesterone priming treatment (treated or not) was or was not associated with a single dose of FSH in a slow-release hyaluronic acid preparation given on Day 0. The follicular population on Day 3 and the number and morphology of recovered cumulus oocyte complexes (COCs) were not affected by the progesterone treatment (P = NS) but were improved by the FSH administration (P < 0.05). An interaction between both treatments was observed (P < 0.05), with more desirable outcome with the females that received both the progesterone and the FSH treatments. In Experiment 2, half of the females received the exogenous progesterone priming, and all females received FSH on Day 0. After follicular aspiration on Day 3, the cleavage rate and the embryo development rate following in vitro fertilization and culture were greater in those females that received the progesterone treatment (P < 0.05). In conclusion, these studies provide evidence that progesterone treatment during follicular growth affects oocyte competence, with the greater progesterone concentrations enhancing the oocyte’s capacity to undergo cleavage and embryo development.

Hormonal stimulation in 4 to 7 months old Nelore (Bos taurus indicus) females improved ovarian follicular responses but not the in vitro embryo production

The inclusion of pre-pubertal bovine females in reproductive management could allow in vitro embryo production and reduce generation interval, thereby causing faster genetic gain of the herd. However, oocytes of pre-pubertal females have lower competence, blastocyst production, and pregnancy rates than those collected from pubertal animals. This study aimed to evaluate the effect of an induced hormonal stimulation on the serum concentrations of Anti-Mullerian hormone (AMH) and FSH, ovarian responses, ovum pick up (OPU), and in vitro produced embryos (IVP) from oocytes obtained from four-to seven-months old Nelore female cattle. In a crossover design, these females were randomly allocated into: 1) Treated Group (TG, n = 9): the animals were subjected to a hormonal protocol (implanted progesterone device, estradiol benzoate, LH, and FSH) from Day 0 (the start of the treatment) to Day 7 (OPU day), and 2) Control Group (CG, n = 9): the females did not receive any hormonal stimulation, but they had ablation of their largest follicles on Day 2 of experiment. Blood collection for serum FSH measurements was done on Days 5, 6, 7, and 8, and collection for serum AMH measurements was done on Days 5 and 8. As hypothesized, TG had higher serum FSH concentrations (p < 0.05) on Day 5 (1.16 ± 0.31 ng/mL), Day 6 (1.21 ± 0.45 ng/mL), and Day 7 (0.95 ± 0.26 ng/mL) than CG (0.56 ± 0.17 ng/mL on Day 5, 0.60 ± 0.25 ng/mL on Day 6, and 0.60 ± 0.14 ng/mL on Day 7). However, serum AMH concentrations were neither significantly different (p > 0.05) between CG and TG, nor between the collection days. Hormonal stimulation also increased (p < 0.05) total follicular population (20.0 ± 4.95 CG vs 26.66 ± 4.24 TG), ovarian diameter (13.08 ± 1.0 mm CG vs 14.81 ± 1.38 mm TG) and number of follicles ≥2.5 mm (6.88 ± 2.14 CG vs 11.55 ± 4.09 TG). In TG, grades I and II oocytes predominated, whereas, in CG grades III and IV oocytes were more abundant (p < 0.05). No significant increases (p > 0.05) in the cleavage (49.33% CG vs 51.42% TG), cleavage > 4 cells (9.33% CG vs 16.19% TG), and blastocysts rates (1.33% CG vs 8.57% TG) were seen in TG. This hormonal protocol increased serum FSH concentrations that possibly contributed to increases in the observed follicle, as well as improving oocyte quality. This exogenous hormonal stimulation increased available oocytes numbers for IVP, despite no increase in the in vitro embryo production efficiency.

Dose and administration protocol for FSH used for ovarian stimulation affect gene expression in sheep cumulus-oocyte complexes.

The present study evaluated the effect of four ovarian stimulation protocols on the follicular population and molecular status of cumulus-oocyte complexes (COCs). Twelve Santa Inês ewes (in a cross-over design) received 80 or 120mg FSH alone in a multiple-dose (MD80 and MD120) regimen or in combination with 300IU equine chorionic gonadotrophin (eCG) in a one-shot (OS80 and OS120) protocol. The follicular population, COC recovery rate, mean COCs per ewe and the rate of brilliant Cresyl blue-positive (BCB+) COCs were similar among treatments (P>0.05). The expression of markers of oocyte competence (ZAR1, zygote arrest 1; MATER, maternal antigen that embryo requires; GDF9, growth differentiation factor 9; BMP15, bone morphogenetic protein 15; Bcl-2, B-cell lymphoma 2; BAX, Bcl-2 associated X protein) and the steroidogenic pathway (ERα, oestrogen receptor α; LHr, LH receptor; FSHr, FSH receptor; STAR, steroidogenic acute regulatory protein) was affected by stimulation. Specifically, the expression of markers of the steroidogenic pathway was reduced with increasing FSH dose in the OS protocol. FSH at a dose of 80mg reduced the expression of FSHr and ERα in the OS versus MD protocol. Conversely, in MD protocol, only LHr was affected by increasing FSH dose. In conclusion, 80mg FSH in the MD or OS protocol was sufficient to promote the development of multiple follicles and obtain fully grown (BCB+) oocytes. The MD protocol may be more appropriate for the production of better-quality oocytes.

Estimation, morphometry and ultrastructure of ovarian preantral follicle population in agouti (Dasyprocta leporina)

ABSTRACT: The aim of this study was to characterize the preantral ovarian follicular population in agoutis (D. leporina) by estimating the number of follicles at each developmental category, and also describe the morphometry and the specific features of the follicle and the oocyte by using light and transmission electron microscopy. The length of each ovary was measured using a caliper rule, longitudinally sectioned into two halves and both were immediately fixed to perform the estimation of follicular population and ultrastructural analysis. The mean (±S.E.M.) population of follicular per pair of ovary was estimated at 4419.8±532.26 and 5397.52±574.91 for right and left ovaries, respectively, but no differences were observed between them. The diameters for follicles, oocyte and nuclei were: 18.62±3.40μm, 12.28±2.37μm and 6.10±0.93μm for primordial, 23.75±5.70μm, 14.22±3.00μm and 6.70±1.24μm for primary and 88.55±17.61μm, 52.85±17.56μm and 22.33±17.61μm for secondary follicles, respectively. The most of the follicles found belonged to the primordial category (86.63%), followed by primary (13.01%) and secondary (0.35%) one. Additionally, polyovular follicles were observed in all the animals and they represented 7.51% of the total follicles counted. The ultrastructural analysis showed that the oocyte presented a central and regular nuclei, displaying a homogenous mass. Among the organelles, the mitochondria were the most abundant and the oocyte Golgi apparatus was rarely observed. In conclusion, this work shows for the first time the characterization of the population of preantral follicles in the ovary of Dasyprocta leporina. Those information will be useful for further development and adaptation of biotechniques such as germplasm cryopreservation and in vitro gametes manipulation.