Reductions in the number of mid-sized antral follicles are associated with markers of premature ovarian senescence in dairy cows

High-producing dairy cows are subfertile; however, the mechanisms responsible for the decreased fertility are unknown. The aim of the present study was to test the hypothesis that culled dairy cows (4\u20138 years old) characterised by \u2018Lo\u2019 ovaries (i.e. those with <10 mid-antral follicles) are affected by premature ovarian senescence. Cows in which both ovaries were \u2018Lo\u2019 ovaries represented 5% of the total population analysed, and exhibited reduced ovarian size (P < 0.001) and increased perifollicular stroma (P < 0.05) compared with age-matched controls (i.e. cows in which both ovaries had >10 mid-antral follicles; \u2018Hi\u2019 ovaries). The total number of follicles, including healthy and atretic primordial, primary, secondary and small antral follicles, was lower in Lo ovaries (P < 0.01). Interestingly, the primordial follicle population in Lo ovaries was lower (P < 0.05) than in the control. Finally, the follicular fluid of mid-antral follicles from Lo ovaries had reduced oestradiol and anti-M\ufcllerian hormone levels (P < 0.05), but increased progesterone concentrations (P < 0.05). Together, these data account for the reduced fertility of cows with Lo ovaries and are in agreement with previous observations that oocytes isolated from Lo ovaries have reduced embryonic developmental competence. Cows with a specific Lo ovary condition may represent a suitable model to address the causes of low fertility in high-yielding dairy cows, as well as the condition of premature ovarian aging in single-ovulating species

Characterization of alpha-Amylase Produced by the Endophytic Strain of Penicillium digitatum in Solid State Fermentation (SSF) and Submerged Fermentation (SmF)

Α-Amylases are enzymes responsible for breaking the α-1.4 bond in polysaccharides with three or more glucose units, occupying the second place in the most widely employed enzymes in industry in the world. The objective of this study was to compare the yields of α-amylase produced by the endophytic fungus, Penicillium digitatum, strain D1-FB, isolated from Baccharis dracunculifolia D.C. (Asteraceae), through the solid state fermentation (SSM) and submerged fermentation (SmF) processes, in addition to characterizing the produced enzyme. The two fermentations were conducted for 120 hours, taking samples every 24 hours to obtain the peaks of production. The enzymes were characterized according to their optimal pH and temperature for performance and stability regarding the incubation in the presence of ions, variations in pH and temperature. The maximum yield of the enzyme was observed with SSF, using rice bran as substrate after 72 hours of fermentation, with 1,625 U/mL. The α-amylase had an optimal pH at 6.5 and optimal temperature at 45°C. All the ions resulted in a decrease in the activity of α-amylase in the concentration of 5mM. The enzyme proved to be quite stable in a pH range of 6.0 to 7.5 and up to the temperature of 37°C, but it presented great drops in activity with temperatures above 45°C and in the presence of ions at the concentration of 5 mM

The endothelial nitric oxide synthase/nitric oxide system is involved in the defective quality of bovine oocytes from low mid-antral follicle count ovaries

In a previous survey concerning cows of reproductive age, we demonstrated that oocytes isolated from ovaries with 10 medium antral follicles (High ovaries, Hi). The aim of the present study was to evaluate whether a defective endothelial nitric oxide synthase/nitric oxide (eNOS/NO) system and vasculature in healthy medium antral follicles is likely to reduce oocyte competence from Lo ovaries. Thus, experiments were conducted to: (1) immunolocalize eNOS protein during folliculogenesis; (2) quantify eNOS protein/vasculature in the follicle wall; and (3) verify if NO donor, S-nitroso acetyl penicillamine (SNAP) administration during in vitro maturation affects developmental competence of oocytes isolated from Lo ovaries. Endothelial-NOS protein was detected in granulosa and theca cells, as well as in blood vessels from primordial to antral follicles. Quantitative analysis indicated that in medium antral follicles from Lo ovaries, eNOS protein expression and vasculature were reduced (P < 0.05). The addition of SNAP improved blastocyst and hatching rates of oocytes from Lo ovaries, promoting a percentage similar to oocytes from Hi ovaries, and reduced the percentage of apoptotic nuclei in in vitro-produced blastocysts (P < 0.05). Results from our study suggest that in bovine ovaries with low mid antral follicle number, a defective eNOS/NO system is related to a reduced follicle vasculature and may impact oocyte quality, thus inducing a premature decline of fertility

Morphological markers to select populations of oocytes with different cultural needs for dedicated pre-maturation protocols

Oocyte’s chromatin gradually becomes more compacted during the final stage of oocyte development and the level of chromatin compaction is considered a marker of oocyte differentiation [Luciano et al, 2014]. Moreover, several studies demonstrate that in vitro pre-maturation treatments (Pre-IVM), aimed to improve the developmental capability of immature oocytes, might behave differently depending on the oocyte metabolic status, when it is isolated from follicle [Luciano et al., 2011]. This study aims at identifying correlations between cumulus-oocyte complex (COC) morphology and oocyte chromatin configuration and secondly at testing the hypothesis that only fully grown oocytes at earlier stages of differentiation with loosely compacted chromatin  (GV1) can benefit from Pre-IVM treatment.   COCs were collected from bovine 2-6mm ovarian follicles, and further divided in three groups according to their morphology (Class-1, 2 and 3) as previously described [Blondin & Sirard, 1995]. Analysis of chromatin configuration revealed that only Class-1 COC was enriched in GV1 oocyte, while Class-2 and 3 presented a similar distribution of GV1, GV2 and GV3 oocytes, where GV2 and 3 oocytes are characterized by increased chromatin compaction [Lodde et al., 2007]. Then COCs were divided into two groups, one containing Class-1 COCs and the other containing Class-2 and 3 COCs and subjected to pre-IVM for 6 hours in presence of cilostamide and 10-4 UI/ml rhFSH. Finally, COCs underwent standard in vitro maturation (IVM) for 22 hours, in vitro fertilization and embryo culture. Blastocyst rate and embryos cell number were assessed at day 7. Pre-IVM positively affected developmental competences of Class-1, while in Classes 2 and 3 Pre-IVM had detrimental effects.In conclusion COCs morphology could be used as a non-invasive approach to select population of oocyte with different cultural needs. These data could be useful in setting-up dedicated IVM protocols considering specific genes and pathways to improve IVP efficiency

Hypoxia as a stimulus upon neonatal swinemeniscus cells: highway to phenotypic maturation of meniscal fibro-chondrocytes?

Menisci are essential structures in the knee joint where they cover fundamental biomechanical and protective roles (1-3). Menisci are characterized by a peculiar structure that, on one hand, allow them to perform their particular role in the stifle joint, but simultaneously make them a very challenging structure to deal with (2). Immature menisci are featured by numerously elongated cells (fibrocytes-like) in a disorganized matrix composed almost completely of collagen type I and few glycosaminoglycans (GAGs) and have a rich vascularization, on the other hand, mature and functional menisci are characterized by few round-shaped cells,a matrix rich of well ordinated collagen fibres (above all collagen type II) and GAGs, and preserve vascularization only in the outer zone (aka red zone) (1). Great interest, in both human and veterinary medicines, is reserved to the treatment of the injuries of the inner and avascular zone (aka white zone) of the meniscus: until now, there are no perfect solutions for the regeneration or the replacement of this tissue once injured (3). This work is focused on the utilization of an environmental factor like hypoxia in meniscal tissue culture, in order to evaluate if it could be utilized to improve meniscal culture with a view to tissue engineering. Ninety menisci from neonatal pigs (day 0) were harvested and cultured under two different atmospheric conditions (hypoxia with 1% O2 and normoxia) until 14 days. Samples were analysed at 0, 7 and 14 days through histochemical (Safranin-O staining), immunofluorescence and RT-PCR (Sox-9, Hif-1a, Hif-2, Collagen I and II, both methods) and biochemical (DNA, GAGs, DNA/GAGs ratio) techniques to record any possible differences in maturation of meniscal cells. Safranin-O staining allowed to show an increment in matrix deposition and round-shape \u201cfibro-chondrocytic\u201d cells quantity of hypoxia-cultured menisci respect to controls under normal atmospheric conditions. The same maturation shifting was observed by means of immunofluorescence and RT-PCR analysis, characterized by an increment of Sox-9 and collagen II, moving from day zero to 14-days under hypoxic environment, and by biochemical analysis,with an increment of DNA/GAGs ratio typical of mature meniscal tissue (characterized by few cells and much GAGs). This study shows that hypoxia can be considered as a booster to achieve meniscal cells maturation and opens considerably opportunities in the field of meniscus tissue engineering. References 1. Dai Z, et al. J Orthop Res 2013 ;31:1514-9, 2. Fox AJS, et al. Clin Anat 2015 ;28:269-87 3. Sosio C, et al. Tissue Eng Part A 2015 ;21:3-4

Ultrastructural and matrix evaluation of morpho-functional age-related changes in dog meniscus

Menisci are essential structures for the knee joint. Different attempts were made trying to replace or regenerate the meniscus after its tear, but the perfect solution is still far away. A better knowledge of the physiologic development of this structure through time could be useful to understand its behavior in the light of the tissue bio-engineering. In this study, the changes in canine meniscal morphology were evaluated to assess how it varies among diverse age stages. The fibers arrangement and matrix deposition in canine menisci from neonatal (died at birth), 10-days, 30-days and adult dogs, dead for causes not related to the present study, were evaluated by means of histochemistry (safranin-O and Sirius red staining), polarized light microscopy, immunofluorescence (collagen I and II) and Scanning Electron Microscopy (SEM). Moreover, quantitative measurements of glycosaminoglycans (GAGs), DNA and GAGs/DNA ratio were performed. The \u201cknotty\u201d structure of neonatal meniscus is probably due to balls of collagen fibres that are not completely stretched until the 30-days stage (Fig 1). The stretching of the fibres starts from the inner portion that is probably the first and the most compressed zone. Safranin-O staining shows how matrix composition vary during growth. Neonatal meniscus is characterized by a huge number of elongated cells (fibroblast-like) and GAGs, features that characterized a still afunctional tissue. With growth, more and more cells assumed a rounded shape. The end-point of the maturation process is represented by the adult meniscus: it is characterized by almost only rounded cells (fibro-chondrocyte-like), in small number, and surrounded by matrix (Fig 1). Nevertheless, 10-30 days interval could be considered the starting point of the meniscus specialization and maturation. Fibres arrangement starts like balls of collagen fibres that follow a disorganized pattern in the neonatal meniscus (Fig 1). In 10-days meniscus, these balls of fibres tend to disappear starting from the meniscus\u2019 inner portion, in association with an initial organization of the fibres according to the longitudinal and radial axes of the meniscus. The organization of fibres network is almost complete at 30-days of life, when all the fibres follow the two main axes of the meniscus and show a well-organized disposition, as seen in adult meniscus. Through the double immunofluorescence it is possible to recognized different aspect of maturation (Fig 3). Neonatal meniscus shows almost only collagen type I fibres. Collagen type I and II co-expression starts at 10 days (yellow) and become more evident in 30-days meniscus in which even a differentiation of the inner and the outer zone starts. The same differentiation persist in adult meniscus that is characterized by a frankly fibro-chondrocitic-like cellular phenotype. Biochemical analysis confirmed that cellularity decrease over the time starting from neonatal to adult (Fig 3). The same decreasing trend is observed in GAGs deposition. Even if 30-days meniscus present a lot of common characteristics with the adult one, the GAGs/DNA ratios show how the latter is the only that present a maturely functional tissue in which a small number of cells is able to produce a matrix rich of GAGs. Meniscal structure changes during growth. The starting point is represented by the neonatal tissue, rich of immature cells and with poor expression of matrix components. The end-point is the adult tissue, characterized by phenotypically mature cells, which assure a functional matrix deposition. Ten-thirty days interval seems to be the turning point of this developmental process. This work highlights how dog meniscal structure changes its morphology among different age stages; this fact may suggest a role of the biomechanical forces, physiologically acting on meniscus, in the development of its ultimate shape and functions. The knowledge of the developmental process of a structure has a capital importance to comprehend its physiologic anatomy and function