pH-dependent effects of procaine on equine gamete activation

Procaine directly triggers pH-dependent cytokinesis in equine oocytes and induces hypermotility in stallion spermatozoa, an important event during capacitation. However, procaine-induced hyperactivated motility is abolished when sperm is washed to remove the procaine prior to sperm-oocyte co-incubation. To understand how procaine exerts its effects, the external Ca2+ and Na+ and weak base activity dependency of procaine-induced hyperactivation in stallion spermatozoa was assessed using computer-assisted sperm analysis. Percoll-washed stallion spermatozoa exposed to Ca2+-depleted (+2 mM EGTA) procaine-supplemented capacitating medium (CM) still demonstrated hyperactivated motility, whereas CM without NaCl or Na+ did not. Both procaine and NH4Cl, another weak base, were shown to trigger a cytoplasmic pH increase (BCECF-acetoxymethyl (AM)), which is primarily induced by a pH rise in acidic cell organelles (Lysosensor green dnd-189), accompanied by hypermotility in stallion sperm. As for procaine, 25 mM NH4Cl also induced oocyte cytokinesis. Interestingly, hyperactivated motility was reliably induced by 2.5-10 mM procaine, whereas a significant cytoplasmic cAMP increase and tail-associated protein tyrosine phosphorylation were only observed at 10 mM. Moreover, 25 mM NH4Cl did not support the latter capacitation characteristics. Additionally, cAMP levels were more than 10x higher in boar than stallion sperm incubated under similar capacitating conditions. Finally, stallion sperm preincubated with 10 mM procaine did not fertilize equine oocytes. In conclusion, 10 mM procaine causes a cytoplasmic and acidic sperm cell organelle pH rise that simultaneously induces hyperactivated motility, increased levels of cAMP and tail-associated protein tyrosine phosphorylation in stallion spermatozoa. However, procaine-induced hypermotility is independent of the cAMP/protein tyrosine phosphorylation pathway

Collection and freezing of equine epididymal spermatozoa

The epididymis and vas deferens store an important number of fertile spermatozoa called the extragonadal sperm reserves. These stored spermatozoa can be collected in an ultimate attempt to preserve viable spermatozoa of a critically ill or dying stallion. Epididymides are collected via routine castration. After cooled transport of the testicles and epididymides, spermatozoa are collected either by retrograde flushing or by the float-up method. Retrograde flushing usually results in a much higher sperm yield and is considered the method of choice. Epididymal spermatozoa can be frozen using standard freezing protocols

Oviductal and uterine leiomyomata in mares

This paper describes a case of a sessile uterine leiomyoma in a 17-year-old chronic infertile Selle Francais mare. The mass was removed by transendoscopic electrocoagulation. In the same period, 725 mares were screened for oviductal and uterine solid masses in a slaughterhouse survey. Two uterine masses and one oviductal mass were detected in three different mares. Histological and immunohistochemical examination revealed leiomyoma in the four masses. To the authors' knowledge, this is the first report of an oviductal leiomyoma in a mare

Fertiliteitsbehandelingen bij het paard: toepassingsmogelijkheden en beperkingen

Recent developments in the assisted reproduction in horses allow to breed foals from sub- and infertile mares, as well as from recently deceased mares or stallions. Oocytes can be obtained from live donor mares by ovum pick-up (OPU), by flushing oocytes from follicles using a transvaginal or transabdominal approach. Post mortem oocytes can be obtained by scraping the follicles. After oocyte maturation, the oocytes can be fertilized in vitro or can be transferred to the oviduct of an inseminated recipient mare (in vivo). Since conventional in vitro fertilization (IVF) is very unsuccessful in the horse, fertilization is performed by intracytoplasmic sperm injection (ICSI). After ICSI, the fertilized oocytes can be transferred to the oviduct of a synchronized recipient mare or further cultured in vitro up to the blastocyst stage. Subsequently, obtained blastocyts can be transferred to the uterus of a recipient mare. In this article, in vitro embryo production in the horse is highlighted, and possible advantages and disadvantages and clinical and scientific applications are reviewed

Update on mammalian sperm capacitation : how much does the horse differ from other species?

In contrast to various other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. In particular, stallion spermatozoa fails to penetrate the zona pellucida, most likely due to incomplete activation of stallion spermatozoa (capacitation) under in vitro conditions. In other mammalian species, specific capacitation triggers have been described; unfortunately, none of these is able to induce full capacitation in stallion spermatozoa. Nevertheless, knowledge of capacitation pathways and their molecular triggers might improve our understanding of capacitation-related events observed in stallion sperm. When sperm cells are exposed to appropriate capacitation triggers, several molecular and biochemical changes should be induced in the sperm plasma membrane and cytoplasm. At the level of the sperm plasma membrane, (1) an increase in membrane fluidity, (2) cholesterol depletion and (3) lipid raft aggregation should occur consecutively; the cytoplasmic changes consist of protein tyrosine phosphorylation and elevated pH, cAMP and Ca2+ concentrations. These capacitation-related events enable the switch from progressive to hyperactivated motility of the sperm cells, and the induction of the acrosome reaction. These final capacitation triggers are indispensable for sperm cells to migrate through the viscous oviductal environment, penetrate the cumulus cells and zona pellucida and, finally, fuse with the oolemma. This review will focus on molecular aspects of sperm capacitation and known triggers in various mammalian species. Similarities and differences with the horse will be highlighted to improve our understanding of equine sperm capacitation/fertilizing events

A stallion spermatozoon's journey through the mare's genital tract: In vivo and in vitro aspects of sperm capacitation

Conventional in vitro fertilization is not efficacious when working with equine gametes. Although stallion spermatozoa bind to the zona pellucida in vitro, these gametes fail to initiate the acrosome reaction in the vicinity of the oocyte and cannot, therefore, penetrate into the perivitelline space. Failure of sperm penetration most likely relates to the absence of optimized in vitro fertilization media containing molecules essential to support stallion sperm capacitation. In vivo, the female reproductive tract, especially the oviductal lumen, provides an environmental milieu that appropriately regulates interactions between the gametes and promotes fertilization. Identifying these 'fertilization supporting factors' would be a great contribution for development of equine in vitro fertilization media. In this review, a description of the current understanding of the interactions stallion spermatozoa undergo during passage through the female genital tract, and related specific molecular changes that occur at the sperm plasma membrane is provided. Understanding these molecular changes may hold essential clues to achieving successful in vitro fertilization with equine gametes

Contralateral Regional Recurrence in Lateralized or Paramedian Early-Stage Oral Cancer Undergoing Sentinel Lymph Node Biopsy-Comparison to a Historic Elective Neck Dissection Cohort

Introduction: Nowadays, two strategies are available for the management of the clinically negative neck in early-stage (cT1-2N0) oral squamous cell carcinoma (OSCC): elective neck dissection (END) and sentinel lymph node biopsy (SLNB). SLNB stages both the ipsilateral and the contralateral neck in early-stage OSCC patients, whereas the contralateral neck is generally not addressed by END in early-stage OSCC not involving the midline. This study compares both incidence and hazard of contralateral regional recurrences (CRR) in those patients who underwent END or SLNB. Materials and Methods: A retrospective multicenter cohort study, including 816 lateralized or paramedian early-stage OSCC patients, staged by either unilateral or bilateral END (n = 365) or SLNB (n = 451). Results: The overall rate of occult contralateral nodal metastasis was 3.7% (30/816); the incidence of CRR was 2.5% (20/816). Patients who underwent END developed CRR during follow-up more often than those who underwent SLNB (3.8 vs. 1.3%; p = 0.018). Moreover, END patients had a higher hazard for developing CRR than SLNB patients (HR = 2.585; p = 0.030). In addition, tumor depth of invasion was predictive for developing CRR (HR = 1.922; p = 0.009). Five-year disease-specific survival in patients with CRR was poor (42%) compared to patients in whom occult contralateral nodal metastases were detected by SLNB or bilateral END (88%), although not statistically different (p = 0.066). Conclusion: Our data suggest that SLNB allows for better control of the contralateral clinically negative neck in patients with lateralized or paramedian early-stage OSCC, compared to END as performed in a clinical setting. The prognosis of those in whom occult contralateral nodal metastases are detected at an earlier stage may be favorable compared to those who eventually develop CRR, which highlights the importance of adequate staging of the contralateral clinically negative neck

Isolation and characterization of functionally active extracellular vesicles from culture medium conditioned by bovine embryos in vitro

Extracellular vesicles (EVs) play a possible role in cell–cell communication and are found in various body fluids and cell conditioned culture media. The aim of this study was to isolate and characterize EVs in culture medium conditioned by bovine embryos in group and to verify if these EVs are functionally active. Initially, ultracentrifuged bovine serum albumin (BSA) containing medium was selected as suitable EV-free embryo culture medium. Next, EVs were isolated from embryo conditioned culture medium by OptiPrepTM density gradient ultracentrifugation. Isolated EVs were characterized by nanoparticle tracking analysis, western blotting, transmission, and immunoelectron microscopy. Bovine embryo-derived EVs were sizing between 25–230 nm with an average concentration of 236.5 ± 1.27 × 108 particles/mL. Moreover, PKH67 EV pre-labeling showed that embryo-secreted EVs were uptaken by zona-intact bovine embryos. Since BSA did not appear to be a contaminating EV source in culture medium, EV functionality was tested in BSA containing medium. Individual embryo culture in BSA medium enriched with EVs derived from conditioned embryo culture medium showed significantly higher blastocyst rates at day 7 and 8 together with a significantly lower apoptotic cell ratio. In conclusion, our study shows that EVs play an important role in inter embryo communication during bovine embryo culture in group

Quantifying the effectiveness of climate change mitigation through forest plantations and carbon sequestration with an integrated land-use model

<p>Abstract</p> <p>Background</p> <p>Carbon plantations are introduced in climate change policy as an option to slow the build-up of atmospheric carbon dioxide (CO₂) concentrations. Here we present a methodology to evaluate the potential effectiveness of carbon plantations. The methodology explicitly considers future long-term land-use change around the world and all relevant carbon (C) fluxes, including all natural fluxes. Both issues have generally been ignored in earlier studies.</p> <p>Results</p> <p>Two different baseline scenarios up to 2100 indicate that uncertainties in future land-use change lead to a near 100% difference in estimates of carbon sequestration potentials. Moreover, social, economic and institutional barriers preventing carbon plantations in natural vegetation areas decrease the physical potential by 75–80% or more.</p> <p>Nevertheless, carbon plantations can still considerably contribute to slowing the increase in the atmospheric CO₂concentration but only in the long term. The most conservative set of assumptions lowers the increase of the atmospheric CO₂concentration in 2100 by a 27 ppm and compensates for 5–7% of the total energy-related CO₂emissions. The net sequestration up to 2020 is limited, given the short-term increased need for agricultural land in most regions and the long period needed to compensate for emissions through the establishment of the plantations. The potential is highest in the tropics, despite projections that most of the agricultural expansion will be in these regions. Plantations in high latitudes as Northern Europe and Northern Russia should only be established if the objective to sequester carbon is combined with other activities.</p> <p>Conclusion</p> <p>Carbon sequestration in plantations can play an important role in mitigating the build-up of atmospheric CO₂. The actual magnitude depends on natural and management factors, social barriers, and the time frame considered. In addition, there are a number of ancillary benefits for local communities and the environment. Carbon plantations are, however, particularly effective in the long term. Furthermore, plantations do not offer the ultimate solution towards stabilizing CO₂concentrations but should be part of a broader package of options with clear energy emission reduction measures.</p