Francesca Ciani

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.

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.

Chronic Administration of Bumetanide Upregulates Calbindin D28k mRNA and Protein Abundance in Rat Distal Convoluted Tubules

Background/Aims: Calbindin D28k has been reported to be involved in transcellular calcium transport along the rat distal convoluted tubule (DCT). It has also been shown that administration of bumetanide is associated with hypercalciuria. The experiments reported here were designed to verify whether chronic infusion of this diuretic affects the gene expression and protein abundance of calbindin D28k along rat kidney DCT. Methods: Bumetanide was subcutaneously infused by an osmotic minipump for 7 days at a rate of 1.5 mg·h–1·kg–1. cDNA was synthesized from total RNA extracted from DCT microdissected from collagenase-treated kidneys. Results: Calbindin D28k mRNA abundance, quantified by competitive PCR, was found to be 13.7 ± 1.9 amol·ng–1 total RNA in DCT of control rats (n = 4) as compared to 24.2 ± 2.4 amol·ng–1 total RNA in DCT of bumetanide- treated rats (n = 5) (p < 0.01). This effect was associated with a 52% increase (p < 0.005) in calbindin D28k protein abundance, as detected by Western blot performed on tissue slices from renal cortex (n = 4). Conclusion: These data not only demonstrate that bumetanide upregulates the mRNA and protein abundance of calbindin D28k in rat DCT, but also suggest that DCT calcium reabsorption is increased following the administration of this loop diuretic.

Anti-proliferative and pro-apoptotic effects of Uncaria tomentosa aqueous extract in squamous carcinoma cells

Uncaria tomentosa (Willd.) DC. (Rubiacee), also known as uña de gato, is a plant that grows wild in the upper Amazon region of Peru and has been widely used in folk medicine to treat several health conditions including cancer. We have produced an aqueous extract from Uncaria tomentosa (UT-ex) and analyzed its effects on squamous carcinoma cells and immortalized HaCaT keratinocytes. Squamous cell carcinoma (SCC) is an uncontrolled growth of abnormal cells arising in the skins squamous layer of epidermis. When detected at an early stage, SCCs are almost curable, however, if left untreated, they can penetrate the underlying tissue and become disfiguring. We have evaluated cell proliferation, apoptosis and the level of reactive oxygen species following UT-ex treatment. UT-ex affected cell cycle progression and reduced cell viability in a dose and time-dependent manner. From a mechanistic point of view, this delay in cell growth coincided with the increase of reactive oxygen species (ROS). Furthermore, PARP1 cleavage was associated to the reduction of Y-box binding protein 1 (YB-1) 36kDa, a nuclear prosurvival factor involved in DNA damage repair. These data indicate that UT-ex-induced cell death can be ascribed, at least in part, to its ability both to induce oxidative DNA damage and antagonize the mechanism of DNA repair relying upon YB-1 activity. They also show that non metastatic SCCs are more susceptible to UT-ex treatment than untransformed keratinocytes supporting the use of UT-ex for the treatment of precancerous and early forms of squamous cell carcinomas. Preliminary chemical investigation of UT-ex revealed the presence of hydrophilic low-medium molecular weight metabolites with anticancer potential towards squamous carcinoma cells.

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.

Influences of dietary supplementation with Lepidium meyenii (Maca) on stallion sperm production and on preservation of sperm quality during storage at 5 °C

Stallion semen is damaged by oxidative stress during cooling and transport. Semen processing and extenders have been tested to improve the fertilizing capacity of semen and to preserve semen during transport. Dietary supplementation with natural antioxidants has been proposed to prevent oxidative damages. In this study, for the first time, the effect of dietary supplementation with Lepidium meyenii (Maca) on the characteristics of fresh and chilled stallion semen was evaluated. Maca is a traditional Andean crop used as a nutraceutical for the fertility‐enhancing properties that are linked with antioxidant activity. The diet of five stallions was supplemented with 20 g of Maca powder daily for a total of 60 days. A control group of five stallions received the same diet without Maca. Semen was collected once before the administration of Maca (D0), twice during the administration at 30 and 60 days (D30 and D60), and finally twice at 30 and 60 days after the end of the administration (D90 and D120). Ejaculates were processed for cooled shipping at 5 °C and evaluated in the laboratory for total and progressive motility, acrosome integrity, and lipid peroxidation after collection and after 24, 48, and 72 h of storage. Dietary supplementation with Maca improved sperm concentration (from 213 ± 80.4 to 447 ± 73.1 × 106 spz/mL) and total sperm count (from 10,880 ± 4377 to 24,783 ± 4419 × 106 spz). The beneficial effects of Maca supplementation on motility and acrosome integrity in the raw semen were detected from the end of treatment with Maca (D60) until the end of the study (D120). Furthermore, during cooling storage, total motility, progressive motility, and acrosome integrity declined more slowly in the Maca‐treated group than in the control group. Lipid peroxidation did not change during cooling storage in either group and did not show a significant difference between the two groups. In this study, the dietary supplementation with Maca increased sperm production and stabilized semen quality during chilled storage.

Influence of γ-glutamyltransferase and alkaline phosphatase activity on in vitro fertilisation of bovine frozen/thawed semen

Abstract The aim of this work was to evaluate whether the residual amount of γ-glutamyl-transferase (GGT) and alkaline phosphatase (ALP) in bovine sperm after freezing/thawing is correlated with fertility parameters, including blastocyst rates after in vitro fertilisation (IVF). The enzyme activities were determined in both spermatozoa and supernatant after centrifugation. While ALP was only correlated with sperm viability, GGT activity was correlated with sperm motility (rs = .4; p < .05) both in sperm and supernatant. Interestingly, GGT activity was also correlated with cleavage (rs = .5; p < .05 and .8; p < .01, for sperm and supernatant respectively) and blastocyst (rs = .6 and .9, for sperm and supernatant respectively; p < .01) rates obtained after IVF. These results suggest that GGT could play an important role in the protection of sperm against oxidative stress and could be considered a reliable marker to assess frozen/thawed sperm quality in bovine.