The luteal outcome of anoestrus ewes induced to ovulate by the male effect is not related to the population of ovarian antral follicles before male exposure.

The ovarian status and its relationship with the response to the male effect were studied in Ile-de-France ewes entering anoestrus early (becoming anovulatory on January-February, n=13) or late (becoming anovulatory on March, n=13). The male effect was performed, in each group of ewes, at the beginning of the anoestrus season (March-April), approximately 35 days after ewes became anovulatory. Transrectal ultrasonography of ovaries was done at D-7, D-5, D-3 and D0 (ram introduction day) to examine the number and size of follicles ≥2mm, from D0 to D4 to analyze the ram-induced preovulatory follicles and at D14-D16 to identify luteal structures. Plasmatic progesterone level was assessed from D-7 to D14-16 to examine the ovulatory response to the male effect. Before ram introduction, the number of medium (3.5-4.4mm) and large (>4.4mm) follicles and the maximum follicle diameter were lower (p<0.05) in ewes entering anoestrus early than in ewes entering it late. The percentage of ewes developing a short cycle at the first ram-induced ovulation was higher in those starting anoestrus early (92% vs 31%; p<0.05); normal cycles were only observed in ewes entering anoestrus late (0% vs 54%; p<0.05). The time of the onset of anoestrus did not affect (p>0.05) the ram-induced preovulatory follicle characteristics; these parameters were similar (p>0.05) between ewes developing a short or a normal cycle. Results did not show any relationship between the ovarian status preceding male introduction and the growing dynamic of the ram-induced preovulatory follicles or the category of cycle (normal or short) displayed following ovulation. In conclusion, (1) the luteal outcome following the first ovulation induced by the male effect depends on the time of onset of seasonal anoestrus and (2) the number and size of follicles ≥2mm also depend on the time of onset of seasonal anoestrus but are not related to the luteal outcome following the first ovulation induced by the male effect.

Anti-Mullerian hormone as a predictive endocrine marker for embryo production in the goat.

Recently, we demonstrated the relationship between anti-Müllerian hormone (AMH) circulating concentrations, ovarian follicles, and embryo production in cattle. However, they have not yet been established in a species with a seasonal breeding activity. Thus, goats were subjected to repeated in vivo embryo production during the breeding season, at the end of the breeding season, and at the end of the anestrus season. Embryo production after FSH treatment was highly repeatable for each goat. Plasma AMH concentrations, measured before the first FSH treatment, were highly correlated with the number of collected, transferable, and freezable embryos, resulting from the three sessions of embryo production. Plasma AMH concentrations transiently decreased after each exogenous FSH treatment, but they showed little change with season, and no relationship was observed between AMH and endogenous FSH concentrations during seasonal transitions. Follicles of 1-5 mm in diameter were the main target of the FSH treatment and were major contributors to circulating AMH concentrations. Granulosa cell AMH expression decreased as the follicle approached terminal development, while the expression of maturation markers (CYP19A1 and FSHR) increased. In conclusion, circulating AMH concentrations can be predictive of the capacity of a donor goat to produce high or low numbers of high-quality embryos. This prediction could be accurately made from a single blood measurement of AMH during either breeding or anestrus seasons. Variability in the number of gonadotropin-responsive follicles of 1-5 mm in diameter between individuals resulted in the differences in circulating AMH concentrations measured between individuals.

In vivo imaging of in situ motility of fresh and liquid stored ram spermatozoa in the ewe genital tract.

The fertility of ram semen after cervical insemination is substantially reduced by 24 h of storage in liquid form. The effects of liquid storage on the transit of ram spermatozoa in the ewe genital tract was investigated using a new procedure allowing direct observation of the spermatozoa in the genital tract. Ejaculated ram spermatozoa were double labeled with R18 and MitoTracker Green FM, and used to inseminate ewes in estrus either cervically through the vagina or laparoscopically into the base of the uterine horns. Four hours after insemination, the spermatozoa were directly observed in situ using fibered confocal fluorescence microscopy in the base, middle and tip of the uterine horns, the utero-tubal junction (UTJ) and the oviduct. The high resolution video images obtained with this technique allowed determination of the distribution of spermatozoa and individual motility in the lumen of the ewe's genital tract. The results showed a gradient of increasing concentration of spermatozoa from the base of the uterus to the UTJ 4 h after intra-uterine insemination into the base of the horns. The UTJ was shown to be a storage region for spermatozoa before their transfer to the oviduct. The in vitro storage of spermatozoa in liquid form decreased their migration through the cervix and reduced the proportion of motile spermatozoa and their straight line velocity at the UTJ and their transit into the oviduct.

Determination of sex and scrapie resistance genotype in preimplantation ovine embryos.

The aim of this study was to test the accuracy of genotype diagnosis after pre-amplification of DNA extracted from biopsies obtained by microblade cutting of ovine embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer to recipients. Sex and PrP genotypes were determined. Sex diagnosis was done by PCR amplification of ZFX/ZFY and SRY sequences after PEP-PCR while PrP genotype determination was performed after specific pre-amplification of specific target including codons 136, 154 and 171. Embryos were collected at Day 7 after oestrus. Blastocysts and expanded blastocysts were biopsied immediately after collection whereas compacted morulae were biopsied after 24 hr of in vitro culture. Eighty-nine biopsied embryos were frozen by vitrification. Fresh and vitrified whole embryos were kept as control. DNA of biopsies was extracted and pre-amplified. Sex diagnosis was efficient for 96.6% of biopsies and PrP genotyping was determined in 95.8% of codons. After embryo transfer, no significant difference was observed in lambing rate between biopsied, vitrified control and fresh embryos (54.5%, 60% and 66.6%, respectively). Embryo survival rate was not different between biopsied and whole vitrified embryos (P = 0.38). At birth, 96.7% of diagnosed sex and 95.4% of predetermined codons were correct. Lamb PrP profiles were in agreement with parental genotype. PEP-PCR coupled with sex diagnosis and nested PCR coupled with PrP genotype predetermination are very accurate techniques to genotype ovine embryo before transfer. These original results allow planning of selection of resistant genotype to scrapie and sex of offspring before transfer of cryopreserved embryo.

Human-derived nanoparticles and vascular response to injury in rabbit carotid arteries: proof of principle.

Self-calcifying, self-replicating nanoparticles have been isolated from calcified human tissues. However, it is unclear if these nanoparticles participate in disease processes. Therefore, this study was designed to preliminarily test the hypothesis that human-derived nanoparticles are causal to arterial disease processes. One carotid artery of 3 kg male rabbits was denuded of endothelium; the contralateral artery remained unoperated as a control. Each rabbit was injected intravenously with either saline, calcified, or decalcified nanoparticles cultured from calcified human arteries or kidney stones. After 35 days, both injured and control arteries were removed for histological examination. Injured arteries from rabbits injected with saline showed minimal, eccentric intimal hyperplasia. Injured arteries from rabbits injected with calcified kidney stone- and arterial-derived nanoparticles occluded, sometimes with canalization. The calcified kidney stone-derived nanoparticles caused calcifications within the occlusion. Responses to injury in rabbits injected with decalcified kidney stone-derived nanoparticles were similar to those observed in saline-injected animals. However, decalcified arterial-derived nanoparticles produced intimal hyperplasia that varied from moderate to occlusion with canalization and calcification. This study offers the first evidence that there may be a causal relationship between human-derived nanoparticles and response to injury including calcification in arteries with damaged endothelium.

Evaluation of renal parenchymal disease in a rat model with magnetic resonance elastography.

Alterations in the mechanical properties or "hardness" of tissues allow physicians to detect disease by palpation. Recently, attempts have been made to quantitate and image these tissue properties with the use of magnetic resonance elastography (MRE). This technique has been validated in ex vivo specimens, including kidney, breast, and prostate. In this study, in vivo MRE imaging of rat renal cortex is demonstrated and validated with a disease model that will facilitate further studies. Normal rats and rats with nephrocalcinosis induced with either 2 or 4 weeks of ethylene glycol exposure were studied with MRE. Histology in the diseased rats documented the presence of nephrocalcinosis. MRE measurements and images of shear stiffness were highly reproducible in individual rats. The shear stiffness of the renal cortex in normal rats was 3.87 kPa (95% CI 2.84-4.90 kPa). The shear stiffness increased to 5.02 kPa (95% CI 3.34-6.70 kPa) after 2 weeks of exposure, and to 6.49 kPa (95% CI 4.84-8.14 kPa) after 4 weeks of exposure (P = 0.0302, alpha < 0.05). MRE is capable of detecting alterations in the tissue mechanical properties of kidneys in vivo. It is a promising noninvasive technique that might have pathologic and prognostic significance.

Evidence of nanobacterial-like structures in calcified human arteries and cardiac valves.

Mechanisms mediating vascular calcification remain incompletely understood. Nanometer scale objects hypothesized to be a type of bacteria (nanobacteria) are associated with calcified geological specimens, human kidney stones, and psammona bodies in ovarian cancer. Experiments were designed to evaluate human vascular tissue for the presence of similar nanometer-scale objects. Calcified human aneurysms (n = 8), carotid plaques (n = 2), femoral arterial plaques (n = 2), and cardiac valves (n = 2) and noncalcified aneurysms from patients with bicuspid aortic valve disease (n = 2) were collected as surgical waste from the Heart Hospital of Austin, Austin, Texas, and Mayo Clinic, Rochester, Minnesota. Whole mounts or adjacent sections from each specimen were examined by electron microscopy, stained for calcium phosphate, or stained with a commercially available antibody (8D10). Filtered (0.2 microm) homogenates of aneurysms were cultured and costained with 8D10 antibody followed by PicoGreen to detect DNA or incubated with [3H]uridine. Staining for calcium phosphate was heterogeneously distributed within all calcified tissues. Immunological staining with 8D10 was also heterogeneously distributed in areas with and without calcium phosphate. Analysis of areas with positive immunostaining identified spheres ranging in size from 30 to 100 nm with a spectral pattern of calcium and phosphorus (high-energy dispersive spectroscopy). Nanosized particles cultured from calcified but not from noncalcified aneurysms were recognized by a DNA-specific dye and incorporated radiolabeled uridine, and, after decalcification, they appeared via electron microscopy to contain cell walls. Therefore, nanometer-scale particles similar to those described as nanobacteria isolated from geological specimens and human kidney stones can be visualized in and cultured from calcified human cardiovascular tissue.