Cryosurvival of ex situ and in situ feline oocytes

Significant advances in feline oocytes cryopreservation have recently been achieved. The oocyte is considered more susceptible to cooling damage than embryos and spermatozoa, however recent results demonstrate that banking of cryopreserved female gametes is an attainable goal. In Felids, as well as in other mammals, oocyte cryopreservation would significantly contribute to the improvement of assisted reproductive technologies aimed to the preservation of biodiversity. Besides the advantages in animal conservation, the use of animal models, as domestic cat, provides the opportunity for further investigations of the principles of oocyte cryobiology which can help to improve current technologies applied to both humans and animals. A review of the literature regarding survival of feline oocytes cryopreserved as isolated cells (ex situ) or enclosed in ovarian follicles (in situ) is provide

Current progress on assisted reproduction in dogs and cats : in vitro embryo production

The objective of the development of assisted reproduction techniques in dogs and cats is their application to non-domestic canine and feline species, most of which are considered threatened or endangered. Among these techniques, an entirely in vitro system for embryo production is effectively an important tool for conservation of wildlife. In the last decade, progress has been made in embryo production in carnivores. It has been shown that canine oocytes can resume meiosis in vitro and that these oocytes can be fertilized and developed in vitro, although at a much lower rate than most other domestic animal oocytes. The reason lies in the dissimilarities of reproductive physiology of the dog compared to other species and the lack of precise information concerning the oviductal environment, in which oocyte maturation, fertilization and early embryonic development take place. Successful in vitro embryo production in the domestic cat has been attained with oocytes matured in vitro, and kittens were born after transfer of IVM/IVF derived embryos. On the basis of these results the in vitro fertilization of oocytes has also been applied in several non-domestic feline species. The effectiveness of such protocols in the preservation of genetic material of rare species can be improved by developing better techniques for long-term storage of gametes. In dogs and cats sperm cells have been successfully frozen and the cryopreservation of oocytes would greatly increase their availability for a range of reproductive technologies. Cryopreserved cat oocytes can be fertilized successfully and their development in vitro after fertilization is enhanced when mature oocytes are frozen. Thus refined techniques of oocyte maturation and fertilization in vitro coupled with oocyte cryopreservation could allow for an easy establishment of genetic combinations when male and female gametes in the desired combination are not simultaneously available, and the propagation of endangered carnivores would be facilitated

Improvement in bovine embryo production in vitro by glutathione-containing culture media

Bovine oocytes were matured, fertilized, and cultured (TCM 199 with serum and co-culture) in vitro (IVMFC) with addition, during different phases of the procedure, of antioxidants: superoxide dismutase (SOD) and reduced glutathione (GSH). The addition of SOD (1,500 or 3,000 IU/ml) did not improve proportions of oocytes undergoing cleavage or the development of embryos to morula and blastocyst stages. The cleavage rates were significantly lower than in the control group (CTR 57.5%) when SOD was present during the insemination interval (IVF) or throughout the entire procedure (IVMFC). Thus when the lower concentration was present for IVF and IVMFC, 35.1% and 36.4% of inseminated oocytes cleaved (P < 0.01 compared to CTR) and cleavage results with the higher concentration during IVF and IVMFC were 38.5% and 29.2% (P < 0.025 and P < 0.001 compared to CTR, respectively). Significant improvements in proportions of oocytes undergoing cleavage (84.5% vs. 57.0%, P < 0.001) and morula/blastocyst development (33.3% vs. 13.9%, P < 0.005) were achieved when GSH (1 mM) was added to the culture medium. In a defined medium for culture (mSOF and BSA) the presence of SOD (3,000 IU/ml) was ineffective, but in a defined medium supplemented with GSH (1 mM) at day 6 postinsemination (i.e., when 90% of developing embryos were in 8-16 cell stages), development to the morula and blastocyst stages was supported for 35.5% of cultured oocytes (P < 0.005 compared to 19.2% for CTR). These data suggest that bovine embryos are sensitive to oxidative stress and that medium supplementation with the radical scavenger glutathione can improve embryo development in vitro

Vitrification of immature feline oocytes with a commercial kit for bovine embryo vitrification

The aim of this study was to evaluate the suitability of a commercial kit for bovine embryo vitrification for cryopreserving cat oocytes and to evaluate comparatively the effects of its use with slow freezing procedure on cryotolerance in terms of morphology and oocyte resumption of meiosis. Germinal vesicle stage oocytes isolated from cat ovaries were either vitrified (n = 72) using a vitrification kit for bovine embryo or slow frozen (n = 69) by exposing oocyte to ethylene glycol solution before being transferred to a programmable embryo freezer. After thawing and warming, oocytes were cultured for 48 h and then were examined for meiosis resumption using bisbenzimide fluorescent staining (Hoechst 33342). Fresh immature oocytes (n = 92) were used as the control group. The proportion of oocytes recovered in a morphologically normal state after thawing \u2044 warming was significantly higher in frozen oocytes (94.5%) than in the vitrified ones (75%, p < 0.01). Morphological integrity after culture was similar in vitrified (73.6%) and slow frozen oocytes (76.8%); however, only 37.5% of the morphologically normal oocytes resumed meiosis after vitrification compared to 60.9% of those submitted to slow freezing procedure (p < 0.01). Fresh oocytes showed higher morphological integrity (91.3%) and meiosis resumption rates (82.6%, p < 0.002) than cryopreserved oocytes, irrespective of the procedure used. These results suggest that immature cat oocytes vitrified with a kit for bovine embryos retain their capacity to resume meiosis after warming and culture, albeit at lower rates than slow frozen oocytes. Vitrification and slow freezing methods show similar proportions of oocytes with normal morphology after culture, which demonstrate that thawed and warmed oocytes that resist to cryodamage have the same chances to maintain their integrity after 48 h of culture

Morphological and acrosomal changes of canine spermatozoa during epididymal transit

Background: During epididymal transit, functional and structural modifications leading to full maturation enable male gametes to reach, recognize and fertilize the oocytes. In dogs, little is known on the modifications of spermatozoa during the passage in the epididymis. The aim of this study was to describe the motility, morphology and acrosomal patterns of canine spermatozoa retrieved from the epididymis caput, corpus and cauda.Results: After the dilution required for the collection of epididymal content, sperm motility was significantly higher(P<0.0001) in the cauda compared to corpus and caput.Proportions of spermatozoa with normal morphology were significantly higher in corpus (P =0.02) and cauda(P<0.0001) compared to caput. Overall morphological abnormalities of the head and neck/midpiece were similar in the three different epididymal regions. A significantly increased prevalence of tail defects, mainly represented by single bent tails, was observed in the corpus compared to caput (P<0.0001) and cauda (P=0.006).Numbers of immature sperm with cytoplasmic droplets decreased from the proximal to the distal region of the epididymis. Particularly, proximal cytoplasmic droplets were more frequently found in spermatozoa collected from the caput epididymis than in the corpus (P<0.0001) and in the cauda (P<0.0001), whereas the occurrence of distal cytoplasmic droplets was higher in the corpus than in the caput (P=0.0003) and in the cauda (P<0.05).Significantly higher proportions of spermatozoa with intact acrosomes were retrieved from the cauda epididymis than from the caput (P=0.03) and the corpus (P =0.008). This difference was mainly due to a lower proportion of spermatozoa with abnormal acrosomes (mainly swollen acrosomes) rather than with absent acrosomes. Conclusions: Canine spermatozoa undergo several modifications in the epididymis. The acquisition of progressive motility, migration of the cytoplasmic droplet and acrosomal reshaping lead to mature spermatozoa which are then stored in the cauda epididymis. From this site, spermatozoa can be retrieved and used in assisted reproductive techniques as a valuable tool for propagating genetic traits of high value individuals that dies accidentally or undergoes orchiectomy for medical purposes. Further investigations should be also focused on the potential use of spermatozoa recovered from other epididymal regions

Identification of bovine doppel protein in testis, ovary and ejaculated spermatozoa

Doppel (Dpl) protein is a recently identified prion-like protein. Although Dpl might be expressed in the brain after prion gene deletion, in both human and mice Dpl is normally expressed only in testis and spermatozoa, where it appears to be involved in male fertility. Little information is available so far about the expression pattern of Dpl in bovines, thus, hampering possible research on the role of this protein in bovine infertility. We have thus, designed, produced and validated through Western blotting a polyclonal antibody against bovine Dpl. With this antibody we then screened bovine tissues for Dpl expression by immunohistochemistry. Ejaculated spermatozoa were screened by flow cytometry and immunocytochemistry. Bovine Dpl was expressed in all the developing stages of germinal cells, from spermatogones to ejaculated spermatozoa, in Sertoli cells and in ovarian follicles (granulosa cells and follicular fluid). Dpl immunoreactivity was also found on other tissues, where endothelial cells, peripheral nerves and scattered lymphocytes stained positive. This distribution pattern suggests that Dpl might be involved in sperm maturation/capacitation in bovines, like it might be in mice. This hypothesis needs to be verified by widespread application of the flow cytometric protocol established in this paper on spermatozoa from animals with reduced fertility

Sox9 Duplications Are a Relevant Cause of Sry-Negative XX Sex Reversal Dogs

Sexual development in mammals is based on a complicated and delicate network of genes and hormones that have to collaborate in a precise manner. The dark side of this pathway is represented by pathological conditions, wherein sexual development does not occur properly either in the XX and the XY background. Among them a conundrum is represented by the XX individuals with at least a partial testis differentiation even in absence of SRY. This particular condition is present in various mammals including the dog. Seven dogs characterized by XX karyotype, absence of SRY gene, and testicular tissue development were analysed by Array-CGH. In two cases the array-CGH analysis detected an interstitial heterozygous duplication of chromosome 9. The duplication contained the SOX9 coding region. In this work we provide for the first time a causative mutation for the XXSR condition in the dog. Moreover this report supports the idea that the dog represents a good animal model for the study of XXSR condition caused by abnormalities in the SOX9 locus

Cat epididymal semen cryopreserved with and without vitamin E: effect on sperm parameters and lipid peroxidation

The aims of this study were to investigate: 1) if the addition of \u3b1-tocopherol (vitamin E) in three concentrations (0.3, 0.6 and 0.9 mM) is able to preserve spermatozoa integrity after thawing and 2) the effect of \u3b1-tocopherol supplementation on lipid peroxidation. Fifty four domestic cats were used in this study constituting 18 pools (3 cats per pool). Each pool was submitted at four experimental groups: group 0 (control) \u2013 epididymal sperm were frozen with a commercial Botucrio\uae extender; group 0.3, group 0.6 and group 0.9 \u2013 the extender was supplemented with 0.3, 0.6 and 0.9 mM of \u3b1-tocopherol, respectively. Each semen sample was evaluated for motility, progressive forward motility, morphology, sperm viability (plasma membrane integrity-PMI), hypo-osmotic swelling test (HOST), before and after thawing. The evaluation of lipid peroxidation reaction by Thiobarbituric Acid Reactive Substances (TBARS) test was performed on thawed semen only. Results demonstrated that there was no significant difference between control and the three \u3b1-tocopherol groups with regards to motility and progressive motility after thawing (P > 0.05). As expected, in fresh samples viability was significantly higher than in all the cryopreserved groups in which there was no positive influence of any of the \u3b1-tocopherol concentration used. Lipid peroxidation was higher in the supplemented groups 0.6 and 0.9 mM of \u3b1-tocopherol than in control and in 0.3 mM group. In conclusion, the addition of \u3b1-tocopherol to the commercial extender had no positive influence on reduction of lipid peroxidation. This topic deserves further investigations to better understand the effect of cryopreservation procedures on epididymal spermatozoa and to establish adequate strategies to counteract sperm cryodamages

In vitro oocyte maturation in Canids: biological and technical pitfalls

The efficiency of oocyte in vitro maturation in Canids is characterized by a low and greatly variable success. The reason may lie in a very peculiar morphological and functional characteristic of Canid oocyte folliculogenesis and maturation. The selection of oocytes which had acquired the ability to undergo meiotic maturation, through adequate intrafollicular growth, is important for in vitro maturation. Moreover, the identification of parameters indicative of meiotic competence could help in the understanding of the mechanisms which limit the maturation rates in these species. In dogs some of these parameters have already been identified. It has been shown that the size of oocytes is important for their ability to mature in vitro and oocytes with a diameter >100 \uf06dm have a better chance to reach the metaphase II (Hewitt and England, Theriogenology 1998, 49:957-966). In addition, it has been reported in the fox that oocytes with diameters up to 100 \uf06dm are meiotically incompetent (Srsen et al., Zygote 1998, 6:299-309). Moreover, advanced preantral and early antral follicles dissected from canine ovaries and cultured in vitro show that their oocytes are competent to resume meiosis to the metaphase II stage (Bolamba et al., Theriogenology 1998, 49: 933-942). Preovulatory maturation is required for the normal development of canine oocytes, although they are ovulated at the germinal vesicle stage. In fact, canine oocytes collected from ovaries before the preovulatory intrafollicular maturation (anoestrous) have lower rates of maturation than oocytes collected from preovulatrory follicles of superovulated bitches (Yamada et al., J. Reprod. Fertil. 1993 Suppl. 47: 227-229). Oocyte and cumulus cell morphology is also important for selecting oocytes. Nickson et al. (J. Reprod. Fertil. 1993 Suppl. 47: 231-240) reported that only oocytes with at least two layers of closely applied cumulus cells are committed to develop in culture. The immature stage of oocytes at ovulation and the persistence of cumulus cells that remain attached in a tight and multilayered mass during the transport and maturation period within the oviduct suggested that the investigation of the communications through gap junctions between the somatic compartment of the follicle and the oocyte, could help to identify competent oocytes. It is well known that such communications are involved in the acquisition of meiotic and developmental competence, and it has been reported in the fox that all junctional contacts between cumulus cells and oocyte are disrupted when metaphase I is reached in vivo (Hyttel et al., Anat Embryol. 1990, 181: 325-331). Moreover, the cumulus, mainly corona radiata cells, controls resumption of meiosis of fox oocytes either in vivo or in vitro conditions (Srsen et al., Zygote 1998, 6:299-309). Recent results (Luvoni et al., J. Reprod. Fertil Suppl., in press) have demonstrated that the functional status of cumulus cells-oocyte communications, through gap junctions, is influenced by the stage of the cycle, and that oocytes collected during late proestrous are capable of completing meiosis at a higher rate than oocytes collected during anoestrous. This suggests that the stage of the cycle at the time of collection influences oocyte meiotic competence. Thus, oocyte diameter, cumulus conformation as well as follicular developmental stage and stage of the cycle are important selection parameters for successful oocyte in vitro maturation in Canids