C. S. Haas

Tretinoin-loaded lipid-core nanocapsules decrease reactive oxygen species levels and improve bovine embryonic development during in vitro oocyte maturation.

In vitro oocyte maturation (IVM) protocols can be improved by adding chemical supplements to the culture media. Tretinoin is considered an important retinoid in embryonic development and its association with lipid-core nanocapsules (TTN-LNC) represents an innovative way of improving its solubility, and chemical stability, and reducing its toxicity. The effects of supplementing IVM medium with TTN-LNC was evaluated by analyzing production of reactive oxygen species (ROS), S36-phosphorilated-p66Shc levels and caspase activity in early embryonic development, and expression of apoptosis and pluripotency genes in blastocysts. The lowest concentration tested (0.25μM) of TTN-LNC generated higher blastocyst rate, lower ROS production and S36-p66Shc amount. Additionally, expression of BAX and SHC1 were lower in both non-encapsulated tretinoin (TTN) and TTN-LNC-treated groups. Nanoencapsulation allowed the use of smaller concentrations of tretinoin to supplement IVM medium thus reducing toxic effects related with its use, decreasing ROS levels and apoptose frequency, and improving the blastocyst rates.

Expression of growth and differentiation Factor 9 and cognate receptors during final follicular growth in cattle

Mutations in growth and differentiation factor 9 (GDF9) gene are associated to sterility or, paradoxically, increased ovulation rate in ewes. Despite its importance, the exact function of GDF9 in ovarian physiology is still poorly understood. This study aimed to investigate GDF9 function during dominant follicle growth and its regulation in follicular fluid. The regulation of GDF9 receptors in GnRH/LH-stimulated granulosa cells was also investigated. In a first experiment, a new follicular wave was induced and the intrafollicular GDF9 treatment into the largest growing follicle (8.5-9.5 mm) at both 100 (n = 3) and 1000ng/ml (n = 4) had no effect on follicular growth, estrus manifestation and ovulation compared to control (PBSinjected) follicles (n = 3). In a second experiment, follicles were obtained just after follicular deviation (day 4 after follicular emergence) and the abundance of GDF9 in follicular fluid did not differ between healthy dominant (n = 4) and atretic subordinate follicles (n = 4), as assessed by western blot analysis. Finally, mRNA expression of BMPR2 and TGFBR1 receptors was evaluated in granulosa cells obtained from preovulatory follicles (>12 mm diameter) obtained 0, 3, 6, 12 or 24 h after i.m. GnRH administration (n = 4-5 follicles/moment). Both receptors were significantly up regulated 12 h after GnRH treatment. Present results do not confirm the hypothesis that GDF9 inhibits dominant follicle growth and suggests a minor role in determining follicle fate. In the other hand, GDF9 receptors regulation in GnRH/LH-stimulated granulosa cells provides the first in vivo evidence of its involvement in the complex cascade of events that culminates in ovulation and luteinization in cattle.

Reproductive nanotechnology: tretinoin-loaded lipid-core nanocapsulesand in vitro embryos production

Background The improvement of in vitro maturation (IVM) protocols through the supplementation with different molecules has become an alternative to increase the culture medium efficiency. Tretinoin (TTN, all-trans retinoic acid, ATRA), is an active metabolite of vitamin A [1], that mediates cell proliferation, cell differentiation, and embryonic development process. In in vitro production embryos systems, TTN acts improving cytoplasmic maturation process in oocytes, developmental competence in early embryos, and quality in blastocysts [2]. Studies have been demonstrated the presence of a, b and g subtypes of retinoic acid receptors (RARa, RARb, RARg) for TTN in oocyte, hatched blastocysts, and cumulus cells [3]. This molecule can also be used for treatment of skin disorders and for anti-tumor treatment, so researchers have been associated TTN with polymeric nanoparticles to protect it from degradation and to improve its chemical stability and efficacy [4]. The aim of present study was to test the concentrationdependent effect of supplementation of free tretinoin (TTN) and tretinoin-loaded lipid-core nanocapsules (TTN-LNC) in bovine in vitro maturation media, and its influence in reactive oxygen species (ROS) production in two-to four-cell stage embryos. In conclusion, tretinoinloaded lipid-core nanocapsules added in in vitro maturation media highly protects embryos at early stage of development against oxidative stress. Methods The experimental groups were established, and cumulus-oocyte complexes (COCs) were matured in oocyte in vitro maturation medium supplemented with 0.25, 0.5 and 1 μM of TTN-LNC or TTN. Control groups of COCs matured without treatment and treated only with blank lipid-core-nanocapsules (LNC) were also examined. The oocytes were in vitro fertilized in order to evaluate the ROS levels in embryos produced by the different treatments. The ROS formation was evaluated in two-to four-cell stage embryos as previously described [5] with some modifications.

Motilidade de espermatozoides bovinos após incubação em fluído folicular

The objective of this work was to evaluate sperm cell motility after intrafollicular artificial insemination (IFAI) in vivo or after incubation in follicular fluid in vitro. In the in vivo experiment, IFAI was performed, followed by the recovery of follicular content 1 to 4 hours later, in order to assess sperm motility. In the in vitro experiment, spermatozoa from a pool of commercial frozen-thawed semen were evaluated for their kinetics after incubation for 1 or 3 hours, either pure (pool, control group) or in follicular fluid (FF). A low motility of sperm cells was observed in the FF samples, both in vitro and in vivo. In vitro, the main parameters negatively affected in the sperm cells incubated in FF, compared with the control, were: total motility (TM), progressive motility (PM), curvilinear distance, and straightness, after 1 hour of incubation; and TM, PM, average path velocity, and curvilinear velocity after 3 hours of incubation. The ovarian follicle and follicular fluid do not provide a suitable environment to maintain bovine sperm cell motility.

Expression of paraoxonase types 1, 2 and 3 in reproductive tissues and activity of paraoxonase type 1 in the serum and seminal plasma of bulls

The paraoxonases types 1, 2 and 3 (PON1, PON2 and PON3, respectively) are enzymes that degrade lipid peroxides, preventing oxidative damages relevant for male reproductive function. This study determined the expression of those three paraoxonases in reproductive tissues of bulls and evaluated correlations among the activity of PON1 in the serum and seminal plasma with breeding soundness parameters in bulls. The expression of PON1, PON2 and PON3 was characterised by RT‐PCR in samples of testicular parenchyma, vesicular glands and epididymis collected from three slaughtered bulls. All three paraoxonases were expressed in the testicular parenchyma, PON2 and PON3 were both expressed in the epididymis head and PON3 was also expressed in the epididymis tail. The PON1 activity was determined in samples of serum and seminal plasma from 110 bulls submitted to breeding soundness evaluation. There was a strong correlation (r = .90) between the activity of the PON1 in both serum and seminal plasma (p < .0001). The PON1 activity in the seminal plasma was positively correlated with ejaculates colour, sperm mass activity (p = .04), motility, vigour and viability (all p < .01). Thus, PON1 may be a potential marker for sperm motility and viability in bulls.