Natascia Cocchia

Cryopreservation of feline epididymal spermatozoa from dead and alive animals and its use in assisted reproduction.

Cryopreservation of gametes is an important tool in assisted reproduction programmes; long-term storage of oocytes or spermatozoa is necessary when in vitro fertilization (IVF) or artificial insemination is to be performed at a future date. Cryopreservation of epididymal spermatozoa offers a potential tool for rescuing genetic material from males of endangered populations. The objectives of this work were to: (1) examine sperm motility, viability, abnormality and acrosome integrity of frozen-thawed domestic cat epididymal spermatozoa; and (2) evaluate the same cryopreservation method on wild feline spermatozoa, needed to preserve their genetic resources. Epididymides were collected from 20 domestic cats during routine neutering procedure and from two wild felines at autopsy. The sperm samples, diluted with 4% glycerol/Tris/egg yolk, were loaded into 0.25 ml mini-straws, exposed to nitrogen vapour and stored in liquid nitrogen. After 4 weeks, samples were thawed and re-evaluated. The quality of each fresh and frozen-thawed sperm sample was tested by determining the motility (54.7 +/- 11.3% and 32 +/- 13.1% respectively for cat spermatozoa; 38.3 +/- 18.7% and 21.5 +/- 16.8% respectively for tiger spermatozoa), viability (74.3 +/- 8.6% and 45.2 +/- 9.4% respectively for cat spermatozoa; 42.4 +/- 14.5% and 33.5 +/- 12.9% respectively for wild felid spermatozoa), morphology and acrosomal status. The present study showed that feline epididymal spermatozoa can be frozen in egg-yolk extender with 4.0% glycerol in 0.25 ml straws. The procedure used in the present study for epididymal cat sperm cryopreservation may be applied to bank the genetic resources of wild felid species.

Comparison of the cytobrush, cottonswab, and low-volume uterine flush techniques to evaluate endometrial cytology for diagnosing endometritis in chronically infertile mares.

Endometritis is the most important cause of infertility in barren mares. The quick method of endometrial cytology (EC) has a relatively high reliability in diagnosing endometrial inflammation in the mare. For reliable cytological results, a collection technique that yields many well-preserved cells representative of a large uterine surface area without causing harm to the reproductive tract is required. The aim of the study was to compare three usually employed techniques for collection of endometrial and inflammatory cells (guarded cotton swab, uterine lavage, and cytobrush) in chronically infertile mares. Twenty Standardbred mares were used. In each mare, samples for EC were collected, first by a cotton swab (DGS), then by a cytobrush (CB), and finally by low volume flush (LVF). The slides were stained using the Diff Quick stain. The following parameters were assessed for each tested technique: background content of the slides; quality of the cells harvested; total cellularity; neutrophils; ratio PMN/uterine epithelial cells; inflammatory cells; vaginal epithelium cells. Categorical variables were compared using contingency tables and Pearson Chi-square tests, whereas continuous variables were compared using one-way analysis of variance (ANOVA); P<0.05 was considered significant. Samplings by DGS and CB resulted easy and quick to perform via a single operator in all cases. LVF was performed easily, but required the presence of 2-3 players and took more time. The background content of the slides prepared by DGS appeared proteinaceous, slides prepared by LVF appeared contaminated by red blood cells or debris, whereas slides prepared by CB appeared clear. All smears showed a good total cellularity. The CB yielded significantly more cells (P<0.0001) than DGS and LVF. The DGS produced significant more cells than LVF (P<0.0001). The DGS produced significantly more (P=0.003) intact cells than CB and LVF. Distorted cells were significantly (P=0.001) more frequent in smears by LVF. The CB harvested significantly (P=0.009) more fragmented cells. CB and LVF produced significantly (P<0.0001; P=0.02) more PMNs/HPF than DGS. In smears collected by LVF the proportion of PMNs/uterine epithelial cells was significantly (P=0.0062; P=0.0023) higher than in smears by CB and DGS. CB collected a significantly higher (P=0.0011) proportion of PMNs than DGS. Acute endometritis was diagnosed in 50% (10/20) of the mares by DGS cytological samples, 25% (5/20) by CB, and 75% (15/20) by LVF. Inflammatory cells other than PMN (lymphocytes, macrophages, eosinophils) were collected exclusively by CB method. Epithelial cells from the vagina were only detected in LVF slides. The agreement of the diagnosis of endometritis between the three techniques of collection and between the different criteria adopted to evaluate smears obtained with the same technique was poor (k≤0.3). In conclusion, results show that cytobrush and flush specimens were superior in all parameters to cotton swab smears. Even though the cytobrush technique requires specialized equipment, sample collection by this method was easier, more consistent, and quicker than the lavage method, indicating that the brush would be the preferred collection method for use on field in the mare. More studies are needed to establish criteria for interpretation of inflammation in the mare on cytobrush samples.

Effect of sod (superoxide dismutase) protein supplementation in semen extenders on motility, viability, acrosome status and ERK (extracellular signal-regulated kinase) protein phosphorylation of chilled stallion spermatozoa.

New studies are underway to find new methods for supporting longer storage of cooled stallion semen. It is known that high concentrations of reactive oxygen species (ROS) cause sperm pathology. The metalloprotein superoxide dismutase (SOD) is responsible for H(2)O(2) and O(2) production, by dismutation of superoxide radicals. The aim of this study is to assess the quality of chilled stallion semen processed with extenders containing SOD at different concentrations as antioxidant additives. A total of 80 ejaculates collected from 5 standardbred stallions was divided into 5 aliquots treated as: native semen (control 1); native semen diluted 1:3 with Kenney semen extender (control 2); spermatozoa diluted after centrifugation in extender without (control 3) or with SOD at 25 IU/ml (experimental 1) or 50 IU/ml (experimental 2). Each sample was analyzed for motility, viability and acrosome status, immediately after semen preparation and again after storage at 5 °C for 24 h, 48 h and 7 2h. Acrosome integrity was evaluated by Chlortetracycline (CTC) and Fluorescent-labeled peanut lectin agglutinin (PNA-FITC conjugated staining). A proteomic approach of quantifying extracellular signal regulated kinase (ERK) was also evaluated as an indirect indicator of oxidative stress. In all samples sperm progressive motility and sperm acrosomal integrity showed a significant reduction between fresh and cooled spermatozoa at 24 h, 48 h and 72 h. Quality parameters of sperm were significantly higher (Progressive Motility P < 0.01; Viability P < 0.001) in aliquots supplemented with SOD. ERK phosphorylation was statistically higher (P < 0.01) in aliquots without SOD. The Authors concluded that addition of SOD to semen extenders improves the quality of chilled equine semen and reduces ERK activation.

Pregnancy detection in mice using ultrasound

DIAGNOSTIC ultrasound has been used in almost all medical fields and is recognised as an increasingly important modality in a variety of clinical situations. The mouse is currently the most widely used animal in biomedical research (Marshall 2000). Diagnosis of pregnancy in experimental mice is possible at 12 to 14 days gestation, when abdominal distension is apparent. Mice with large litters may show distension slightly earlier (Kaufman 1989). Diagnosis of pregnancy at 7·5 days in experimental mice is currently performed by direct inspection of the uterine horns after laparotomy, with no routine utilisation of imaging techniques. The embryonic mouse is a ubiquitous model of mammalian development, due to the obvious benefits of a short gestation cycle, and genetic homologies with developmental genes in other mammals. Such models should yield further insight into the mechanisms responsible for human developmental and disease processes. Most previous investigations of the gestation period in the mouse have been invasive in nature. Video microscopy has been used to monitor heart dynamics in surgically exposed embryos, magnetic resonance microscopy has been successfully utilised to visualise threedimensional anatomy and vasculature in fixed mouse embryos (Smith and others 1994), and servo-null pressure measurement and Doppler interrogation using implanted crystals have also been used (Keller and others 1996, MacLennan and Keller 1999). However, none of the previous methods used have provided real-time imaging of live embryos as early as 7·5 days. The lack of effective in utero imaging methods has been a significant limitation. This short communication describes the capabilities of a conventional ultrasound system as a tool for the noninvasive investigation and diagnosis of early pregnancy in the mouse. All the animals in the present study were maintained according to protocols approved by the Institutional Animal Research and Care Committee, University of Naples. Fifty-one pregnant CD1 mice, ranging in age from seven to nine weeks, were used in the study. The mice were divided into three groups of 17 mice at three different stages of pregnancy (7·5 days, 12·5 days and 16·5 days). In staging the embryos, gestational day 0·5 was defined as noon of the day a vaginal plug was found following overnight mating. FIG 1: Longitudinal section of the gestational sac (arrows) and placenta (arrowheads) at 7·5 days. The image to the left shows the full urinary bladder

Sex determining of cat embryo and some feline species

Sex identification in mammalian preimplantation embryos is a technique that is used currently for development of the embryo transfer industry for zootechnical animals and is, therefore, a resource for biodiversity preservation. The aim of the present study was to establish a rapid and reliable method for the sexing of preimplantation embryos in domestic cats. Here we describe the use of nested PCR identify Y chromosome-linked markers when starting from small amounts of DNA and test the method for the purpose of sexing different species of wild felids. To evaluate the efficiency of the primers, PCR analysis were performed first in blood samples of sex-known domestic cats. Cat embryos were produced both in vitro and in vivo and the blastocysts were biopsied. A Magnetic Resin System was used to capture a consistent amount of DNA from embryo biopsy and wild felid hairs. The results from nested PCR applied on cat blood that corresponded to the phenotypical sex. Nested PCR was also applied to 37 embryo biopsies and the final result was: 21 males and 16 females. Furthermore, beta-actin was amplified in each sample, as a positive control for DNA presence. Subsequently, nested PCR was performed on blood and hair samples from some wild felines and again the genotyping results and phenotype sex corresponded. The data show that this method is a rapid and repeatable option for sex determination in domestic cat embryos and some wild felids and that a small amount of cells is sufficient to obtain a reliable result. This technique, therefore, affords investigators a new approach that they can insert in the safeguard programmes of felida biodiversity.

Assessment of dual-energy x-ray absorptiometry for use in evaluating the effects of dietary and environmental management on Hermann's tortoises (Testudo hermanni)

OBJECTIVE To assess dual-energy x-ray absorptiometry (DXA) for evaluating effects of diet and environment on bone mineral density in Hermanns tortoises (Testudo hermanni). ANIMALS 26 Hermanns tortoises within 1 month after hatching. PROCEDURES Group 1 was housed in an artificial setting and fed naturally growing vegetation. Group 2 was housed in an artificial setting and fed vegetables grown for human consumption. Group 3 was maintained in an outside enclosure and fed naturally growing vegetation. After 10 months, pyramidal growth, body weight, and adverse conditions were assessed. Bone mineral density (BMD) of the axial and appendicular skeleton, shell, vertebral column, and pelvis was measured via DXA. RESULTS Group 2 had the highest mean ± SD body weight (65.42 ± 30.85 g), followed by group 1 (51.08 ± 22.92 g) and group 3 (35.74 ± 7.13 g). Mean BMD of the shell varied significantly among groups (group 1, 0.05 ± 0.03 g/cm(2)•m; group 2, 0.09 ± 0.15 g/cm(2)•m; and group 3, undetectable). The BMD of the axial and appendicular skeleton, vertebral column, and pelvis did not differ significantly among groups. Pyramidal growth was highest in group 1 and not evident in group 3. CONCLUSIONS AND CLINICAL RELEVANCE Tortoises raised in artificial conditions did not have deficits in BMD, compared with results for outdoor-housed hibernating tortoises. Supplemental calcium was apparently not necessary when an adequate photothermal habitat and plant-based diet were provided. Higher BMD of captive-raised tortoises was morphologically associated with a higher incidence of pyramidal growth in captive-raised groups.

The effects of superoxide dismutase addition to the transport medium on cumulus–oocyte complex apoptosis and IVF outcome in cats (Felis catus)

The aim of the present study was to examine the effects of superoxide dismutase (SOD) addition to the ovary transport medium (4°C, 3-72 h) on ovarian cell viability and apoptosis and in vitro embryo production (IVEP) in domestic cats. The ovaries collected from 76 mixed-breed domestic queens were randomly assigned to the control or SOD-treated groups and incubated for 3, 24, 48 or 72 h. The ovaries were then subjected to the following: (1) fixed in formalin to assess the incidence of apoptosis (fragmented DNA in situ detection kit), (2) stored at -196°C in liquid nitrogen to evaluate the expression of the pro-apoptotic Bax gene and the anti-apoptotic Bcl-2 gene (RT-PCR), and (3) used to obtain the cumulus-oocyte complexes (COCs) in order to test the cell viability (carboxyfluorescein or trypan blue staining) and IVEP. The incidence of apoptosis appeared to be higher in the control compared with the SOD-treated ovaries. The ovarian expression of Bax was lower and the Bcl-2 expression was higher in the SOD-treated group compared with the control group. The presence of SOD in the transport medium increased the viability of COCs and IVEP compared with the control medium. In summary, the supplementation of the ovary transport medium with SOD reduced cellular apoptosis and enhanced COC survival and IVEP in domestic cats.

Reactive Oxygen Species (ROS) and Male Fertility

Oxidative energy production is inevitably associated with the generation of reactive oxygen species (ROS), excessive concentrations of which can lead to cellular pathol‐ ogy. A free radical may be defined as any molecule that has one or more unpaired electrons. The superoxide anion, the hydroxyl radical, and the hypochlorite radical are some of the highest reactive radicals of oxygen. Owing to their high reactivity and to their capability of initiating an uncontrolled cascade of chain reactions, ROS produce extensive protein damage and cytoskeletal modifications and inhibit cellular mechanisms. Aerobic organisms are equipped with a powerful battery of mechanisms that protect them from the adverse effects of lipid peroxidation (LPO) and other manifestations of oxygen toxicity. Defective sperm function frequently causes male infertility, due to abnormal flagella movement, failure to recognize the zona, and inhibition of sperm-oocyte fusion. ROS are fundamental mediators of physiological sperm function, such as signal transduction mechanisms that have an effect on fertility. ROS can have positive effects on sperm and the concentration functions depending on the nature and the concentration of the ROS involved. They are necessary in regulating the hyperactivation and the ability of the spermatozoa to undergo acrosome reaction. An increased amount of superoxide anion (O2) is one of the first steps required by the spermatozoa for induction and development of hyperactivation and capacitation. Numerous studies have shown that oxidative stress plays an important role in the pathophysiology of infertility and assisted fertility. The paternal genome is of primary importance in the normal embryo and fetal develop‐ ment. ROS-induced sperm damage during sperm translation, such as signal trans‐ duction through the seminiferous tubules and epididymis, is one of the most important mechanisms leading to sperm DNA damage. Male germ cells are extremely

Influence of ROS on Ovarian Functions

High level of ROS (Reactive Oxygen Species), due to an increased production of oxidant species and/or a decreased efficacy of antioxidant system, can lead to oxidative stress (OS) an emerging health risk factor involved in the aging and in many diseases, either in humans or in animals. ROS are a double-edged sword – they serve as key signal molecules in physiological processes, but also have a role in pathological processes involving the female reproductive tract. ROS affect multiple physiological processes in reproduction and fertility, from oocyte maturation to fertilization, embryo development and pregnancy. Several studies indicate that follicular atresia in mammalian species due to the accumulation of toxic metabolites often results from oxidative stress. It has been suggested that ROS under moderate concentrations play a role in signal transduction processes involved in growth and protection from apoptosis. Conversely, increase of ROS levels is primarily responsible for the alteration of macromolecules, such as lipids, proteins and nucleic acids, that lead to significant damage of cell structures and thereby cause oxidative stress. To prevent damage due to ROS, cells possess a number of non-enzymatic and enzymatic antioxidants. Non-enzymatic antioxidants include vitamin C, glutathione and vitamin E. Enzymatic antioxidants consist of superoxide dismutases (MnSOD and Cu/ZnSOD) that convert superoxide into hydrogen peroxide; glutathione peroxidase (GPX) and catalase (CAT) which neutralize hydrogen peroxide. Intracellular homeostasis is ensured by the complex interaction between pro-oxidants and

Effect of superoxide dismutase, catalase and glutathione peroxidase supplementation in the extender on chilled semen of fertile and hypofertile dogs

This study investigated the correlation between oxidative stress status and key canine sperm parameters and the effect of addition of a superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) combination in egg yolk tris-citrate glucose (EYT-G) extender on semen during 10 days of storage at 4℃. Ten Boxer dogs were divided into two groups, fertile (F) and hypofertile (H), depending on pregnancy and live birth rate status in the previous year. Semen evaluation was performed on the day of collection (D0) and after 5 (D5) and 10 (D10) days of cooled storage. Sperm motility, kinetic parameters, and DNA integrity were assessed. A correlation between oxidative status and key semen parameters in both F and H groups was observed. Total and progressive motilities were significantly higher in the treated (SOD, CAT, and GPx addition) versus control groups at D10 in both F and H groups, and at D5 in the H group. DNA integrity was significantly higher in both treated groups (H and F) at D5 and D10. In conclusion, the addition of SOD, CAT, and GPx in the extender allows preservation of semen quality for up to 10 days of storage at 4℃ in both fertile and hypofertile dogs.