Accuracy of ultrasound and visual inspection in antral follicular count in crossbred Holstein cows raised under grazing systems at high altitude.

The objective of this research was to investigate the diagnostic accuracy of post-mortem ultrasound in antral follicle count (AFC) determination and compare it with visual AFC in grazing crossbred Holstein cows, at high altitude in Ecuador. Pre-mortem blood from 80 cows was collected, and AFC and ovarian characteristics were analysed post-mortem by ultrasound and visual techniques. AFC counts were stratified as high, medium or low by terciles. Mean AMH concentration in pre-mortem blood was 280.1 ± 15.53 pg/mL. The AFC obtained by visual inspection (26.9 ± 9.49 follicles) was 23.8% higher than by ultrasound (20.5 ± 7.53 follicles) in all ovaries. Body condition score, age and weight of the cattle did not interact with the count technique. In the low AFC group, visual inspection and ultrasound provided similar AFC results. However, in the Medium- and High-AFC groups, AFC by ultrasound was 14.9% lower than AFC by visual inspection. We confirm that ultrasound can be used with great accuracy for AFC >3 mm (close to the resolution limit) in grazing crossbred Holstein cows at high altitude.

Altrenogest treatment during the last week of lactation on ovarian traits and subsequent reproductive performance of primiparous and multiparous sows.

High-quality follicles result in larger corpora lutea (CL), producing more progesterone, and having a fundamental role in pregnancy maintenance. For some sows, follicular growth takes place during lactation, and follicle selection occurs under a catabolic environment. As altrenogest inhibits follicular development, this study aimed to evaluate follicular growth, CL size, estrus expression, and subsequent reproductive performance of sows treated with altrenogest during the last seven days of a three-week lactation. A total of 81 primiparous and 319 multiparous sows were allocated to two treatments: CONT (control group) and ALT (20 mg of altrenogest/day during the last seven days of lactation). Subsamples of 20 primiparous sows and 97 multiparous were randomly selected to evaluate follicular growth and 26 multiparous sows for serum progesterone analysis at day 21 of gestation. On day 21 of pregnancy, CL measurement was performed by ultrasound. Once in estrus, sows were post-cervically inseminated with pooled semen doses with 1.5 × 109 sperm cells at estrus onset and every 24 h during the standing reflex period. Sows not showing estrus until 10 days after weaning were considered in anestrus. The variables weaning-to-estrus interval, CL size, litter size in the subsequent cycle, and piglet birth weight were evaluated using the GLIMMIX procedure and compared using the Tukey-Kramer test. Anestrus, pregnancy, farrowing, and adjusted farrowing rate were evaluated as binary responses using logistic regression. Follicular size was analyzed as a repeated measure during treatment and after weaning. Treatment was considered as a fixed effect. During the treatment period, follicular size was smaller in ALT sows than CONT sows (3.29 vs. 3.52 mm; P < 0.001). However, after treatment, ALT sows showed a larger follicular size than CONT sows (5.30 vs. 5.03 mm; P ≤ 0.01). There were less ALT sows showing estrus than CONT sows on days three (1.03 vs. 4.57%) and four (55.38 vs. 68.02%) after weaning (P ≤ 0.05), respectively. At 21 days after insemination, ALT sows showed larger CL size and lower CL size variation (P < 0.01) than CONT sows. Anestrus rate, pregnancy rate, farrowing rate, adjusted farrowing rate, litter size in the subsequent cycle, piglet birth weight, litter birth weight, and birth weight variation did not differ between treatments (P ≥ 0.14). In conclusion, altrenogest treatment during the last week of lactation concentrated estrus expression on day five after weaning, larger follicle and CL sizes; however, with no improvement in reproductive performance.

Intrafollicular injection of nonesterified fatty acids impaired dominant follicle growth in cattle.

Dairy cows frequently undergo a state of negative energy balance (NEB) after parturition and some have impaired ovarian functions that result in delayed resumption of estrous cyclicity and development of follicles without ovulation occurring. During the postpartum period, cows undergo body-fat store losses, hormonal changes, fat mobilization and increases in nonesterified fatty acid (NEFAs) concentrations in blood and follicular fluid. The effect of NEFAs on follicular development and function of follicular cells, however, is not fully understood. The aim of this study, therefore, was to study the effect of an intrafollicular injection of a mixture of oleic, stearic and palmitic NEFAs on dominant follicle development and function of granulosa cells in cows that were not in a NEB state. Follicular size was less at 24 and 48 h after administration of NEFAs compared to that of control follicles injected with vehicle only. At 24 h after intrafollicular injection, the relative mRNA transcript abundance for proteins involved in steroidogenesis (CYP19A1, 3BHSD, STAR, FSHR), metabolism (GLUT1, GLUT3, INSR, IRS1, IRS2, SLC27A1, PPARG), and cell proliferation and apoptosis (CCND2; XIAP) in granulosa cells, as well as estradiol concentrations in follicular fluid were similar in control and NEFA-treated follicles. In conclusion, the results of this study indicate increased intrafollicular concentrations of NEFAs in cows that are not in a NEB state has a detrimental effect on follicle development. We propose intrafollicular injection is a useful approach to further investigate the local effects of NEFAs on the function of follicular cells.

Methods to study ovarian function in monovulatory species using the cow as a model

In this review, we discuss the utility of the cow as an in vivo model to study the regulation of ovarian functions in monovular species. It is increasingly becoming clear that besides endocrine control locally produced factors play pivotal roles during dominant follicle selection, oocyte maturation, ovulation and luteolysis. Although in vitro culture systems have been used to study these processes, definitive understanding the interactions between endocrine and local factors requires appropriate in vivo models. Most of the experimental approaches to study ovarian functionsin vivo in large animals are based on the use of ultrasonography and considerable progress in this field has been made during the last thirty years. It has been shown that cows are an excellent model to collect samples (e.g., follicular fluid, granulosa cells, oocytes) from live animals at specific stages of follicular development in order to study mechanisms of intrafollicular factors in a physiological endocrine environment. In addition to support fundamental studies, the cow model has contributed immensely to the refinement of assisted reproductive technologies, which are now widely used not only in farm animals but also in humans.

Methods to study ovarian function in monovulatory species using the cow as a model

In this review, we discuss the utility of the cow as an in vivo model to study the regulation of ovarian functions in monovular species. It is increasingly becoming clear that besides endocrine control locally produced factors play pivotal roles during dominant follicle selection, oocyte maturation, ovulation and luteolysis. Although in vitro culture systems have been used to study these processes, definitive understanding the interactions between endocrine and local factors requires appropriate in vivo models. Most of the experimental approaches to study ovarian functionsin vivo in large animals are based on the use of ultrasonography and considerable progress in this field has been made during the last thirty years. It has been shown that cows are an excellent model to collect samples (e.g., follicular fluid, granulosa cells, oocytes) from live animals at specific stages of follicular development in order to study mechanisms of intrafollicular factors in a physiological endocrine environment. In addition to support fundamental studies, the cow model has contributed immensely to the refinement of assisted reproductive technologies, which are now widely used not only in farm animals but also in humans.(AU)

Effect of miR-125b on dermal papilla cells of goat secondary hair follicle

Background: MicroRNAs (miRNAs) are endogenous noncoding RNAs that regulate various biological processes. miR-125b is a miRNA that has been reported to be critical for hair follicle (HF) morphogenesis and development. We identified that the expression of miR-125b varies during an individual hair cycle (anagen, catagen, and telogen) in the skin of cashmere goats. We constructed a gain model (by overexpressing miR-125b) and a loss model (by inhibiting endogenous miR-125b) based on dermal papilla cells (DPCs) to further investigate the role of miR-125b in HF cycle. In addition, we used a dual-luciferase system to highlight the predicated target genes of miR-125b. Results: We found that miR-125b affects the expression of FGF5, IGF-1, SHH, TNF-α, MSX2, LEF-1, FGF7, NOGGIN, BMP2, BMP4, TGF-ß1, and ß-catenin. The dual-luciferase assay further validated a direct interaction between miR-125b and FGF5 and TNF-α. Conclusion: miR-125b affects the expression levels of genes related to hair cycle and may also play a critical role in regulating the periodic development of HF.

Tissue localization of GM-CSF receptor in bovine ovarian follicles and its role on glucose uptake by mural granulosa cells.

The granulocyte-macrophage colony stimulating factor (GM-CSF) is a multifunctional cytokine implicated in proliferation, differentiation, and activation of several cell types including those involved in hematopoiesis and reproduction. In the present study, the expression of the α- and ß-subunit genes of GM-CSF receptor during follicular development in cattle was assessed. The spatial association of α- and ß-subunits of GM-CSF with follicle stimulating hormone receptor (FSHR) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and the temporal associations with gene expression of hexose transporters (GLUTs) in granulosa cells of cattle were also evaluated. The effect of GM-CSF on the functionality of hexose transporters was also determined in an in vitro primary culture of granulosa cells. The spatial association of subunits of the GM-CSF receptor with 3ß-HSD and FSHR suggests a potential steroidogenic regulation of GM-CSF in granulosa cells. Immunodetection of GLUTs and uptake kinetic assays confirmed expression and functionality of these genes for hexose transporters in granulosa cells of cattle. Treatment of granulosa cells with GM-CSF, FSH or insulin- like growth factor-I (IGF-I) alone increased 2-deoxyglucose (DOG) or 3-0-methylglucose (OMG) uptake; however, when cells were treated with various combination of these factors there were no additive effect. Unexpectedly, the combination of GM-CSF and FSH decreased DOG uptake compared to FSH treatment alone. Thus, the expression pattern of GM-CSF receptor subunit genes during follicle development in cattle and promotion of DOG and OMG uptake in granulosa cells indicate a role for GM-CSF, FSH and/or IGF-I alone in regulating granulosa cell metabolic activity, specifically by promoting glucose uptake.

Inhibition of serotonin reuptake in the prepubertal rat ovary by fluoxetine and effects on ovarian functions.

Fluoxetine (FLX), a selective serotonin reuptake inhibitor is an antidepressant in the treatment of mood disorders. Its impact on reproductive processes is incompletely known. The present study analyzed the reproductive effects of FLX in prepubertal female rats. Two experiments were conducted. First (acute administration), 30-day-old female rats were injected intraperitoneally with 5mg/kg of fluoxetine-hydrochloride, and were terminated 24, 48 or 72h after the treatment. Second (subchronic administration), FLX was injected on days 30-33 of age, and the animals were terminated the day of first estrus. In acute treatment estradiol concentration increased to 72h. In subchronic treatment increased serotonin concentration in ovaries and decreased the number of ova shed. An increase in number of atretic follicles and oocyte fragmentation was observed in these animals. The results suggest that FLX acts on the ovary or hypothalamus-pituitary axis resulting in modifications of the follicular development and ovulation.

Three-dimensional modeling of color Doppler images: a new approach to study follicular vascularization in cattle

Ovarian blood supply is directly related to follicle developmental potential and to oocyte quality, and color Doppler ultrasonography might be a valuable tool to predict in vitro fertilization outcomes. In most studies in large domestic animals, however, the evaluation of follicle blood flow is qualitative (presence or absence of color signal) or dependent on the analysis of a single image. The objective of the present study was to first describe the use of a three-dimensional (3D) modeling of color Doppler images for a quantitative assessment of vascularization in bovine ovarian follicles. Follicular wave emergence was synchronized in Holstein and Gir heifers (n = 20), and follicular dynamics were assessed every 12 h using a color Doppler ultrasound device. The recorded cine-loop of the dominant follicle was decomposed into frames and medical image processing software was used to isolate the Doppler signal, generate the 3D model and calculate the volume of vascularization. In experiment 1, the model was validated by comparing the expected and calculated volumes and was used to predict possible variations in the results of the 2D approach. In experiment 2, vascularization was analyzed during follicular development. In both breeds, the volume of vascularization increased after follicle deviation and was positively correlated (P < 0.05) to follicular diameter (r = 0.65 and 0.54 for Holstein and Gir heifers, respectively). Spatial analysis of the three-dimensional model showed an uneven distribution of vascularization in the follicular wall, with a more intense blood flow being detected in the basal (nearest the ovarian hilus) and lateral regions of the dominant follicles. These results demonstrate the potential of this technique as a new tool for in vivo studies of ovarian physiology in large animals.

Three-dimensional modeling of color Doppler images: a new approach to study follicular vascularization in cattle

Ovarian blood supply is directly related to follicle developmental potential and to oocyte quality, and color Doppler ultrasonography might be a valuable tool to predict in vitro fertilization outcomes. In most studies in large domestic animals, however, the evaluation of follicle blood flow is qualitative (presence or absence of color signal) or dependent on the analysis of a single image. The objective of the present study was to first describe the use of a three-dimensional (3D) modeling of color Doppler images for a quantitative assessment of vascularization in bovine ovarian follicles. Follicular wave emergence was synchronized in Holstein and Gir heifers (n = 20), and follicular dynamics were assessed every 12 h using a color Doppler ultrasound device. The recorded cine-loop of the dominant follicle was decomposed into frames and medical image processing software was used to isolate the Doppler signal, generate the 3D model and calculate the volume of vascularization. In experiment 1, the model was validated by comparing the expected and calculated volumes and was used to predict possible variations in the results of the 2D approach. In experiment 2, vascularization was analyzed during follicular development. In both breeds, the volume of vascularization increased after follicle deviation and was positively correlated (P < 0.05) to follicular diameter (r = 0.65 and 0.54 for Holstein and Gir heifers, respectively). Spatial analysis of the three-dimensional model showed an uneven distribution of vascularization in the follicular wall, with a more intense blood flow being detected in the basal (nearest the ovarian hilus) and lateral regions of the dominant follicles. These results demonstrate the potential of this technique as a new tool for in vivo studies of ovarian physiology in large animals.(AU)