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The article reviews the main levels of hormonal regulation of sexual activity in domestic cats, emphasizing the peculiarities that clearly differentiate this species of canids. In the second part, current data on the control of reproduction in this species are presented, emphasizing the treatments based on modern molecules, with low side effects and a wide range of applications. [ABSTRACT FROM AUTHOR]

Diagnostic imaging is one of the most important tools in the breeding soundness evaluation of dogs and cats with reproduction problems. In recent years several imaging techniques have been developed, trying to aid the diagnosis and to differentiate between abnormal findings. This review presents the current knowledge on the imaging of normal and abnormal testes, spermatic cord, excurrent duct system, scrotum, accessory sex glands, penis and muscles for protrusion, erection and ejaculation of the dog and of cat. It also highlights the weak points and disadvantages of each imaging technique. • This review presents the current knowledge on the imaging of normal and abnormal male genital tract of the dog and cat. • It highlights the weak points and disadvantages of each imaging technique. • It can be the starting point for further research on the imaging techiniques of the male reproductive tract of small animals. [ABSTRACT FROM AUTHOR]

The anti‐Müllerian hormone (AMH) is a glycoprotein secreted by Sertoli cells in males and granulosa cells in females. It has first been determined in blood serum of dogs and cats by Place et al. in 2011 with the use of a human‐based ELISA test. Meanwhile, different immunoassays have been validated for AMH determination in animals and a variety of studies have demonstrated the clinical significance of AMH. This review summarizes the current knowledge about AMH in dogs and cats and describes future opportunities for its diagnostic use. [ABSTRACT FROM AUTHOR]

The article offers information about the discovery of first active breeding den of Chinese mountain cat, endangered felid species in the world and endemic to the eastern edge of the Tibetan Plateau, China. It discusses the Chinese mountain cat behaviors, along with information that Chinese mountain cat assessed as Vulnerable by the IUCN Red List.

Abstract Equine chorionic gonadotropin (eCG) has been commonly used to induce estrus in several felid species. However, the mechanisms by which this gonadotropin regulates cat folliculogenesis are still unclear. We investigated the responsiveness of cat ovarian follicles at different developmental stages to various eCG concentrations supplemented in vitro. Follicles were mechanically isolated from the ovaries of 22 cats and categorized into three developmental stages based on their morphology and diameter: 1) two-layered secondary follicle (SF), 100–150 μm (n = 139); 2) multi-layered SF, 150–300 μm (n = 154); and 3) early antral follicle (AF), ≥300–500 μm (n = 123). The follicles were then encapsulated in 0.5% (w/v) sodium alginate and cultured for 12 days in Minimum Essential Medium supplemented with 0, 0.05, 0.1 or 0.5 IU/mL eCG. Follicle and oocyte diameters were assessed every 3 days. On Day 12, mRNA expression levels of FHSR, LHCGR, GDF9, BMP15, CYP17A1, CYP19A1 and STAR were analyzed using real-time PCR. After being cultured for 12 days, follicle growth and mRNA expression of two-layered SF were not influenced by eCG at all concentrations (P > 0.05). However, the concentration of eCG at 0.05 IU/mL stimulated follicular growth and gene expressions in the multi-layered SF and early AF (P < 0.05). Correspondingly, the diameter of oocytes in the multi-layered SF and early AF treated with 0.05 IU/mL eCG was unchanged. Considering the mRNA expression, the level of STAR was enhanced in the early AF (P < 0.05) and tended to increase in the multi-layered SF (P = 0.08) cultured in 0.05 IU/mL eCG, whereas the expression of other genes was not affected. In sum, the responsiveness of cat follicles to eCG is apparent from the multi-layered SF stage onward. The eCG supplementation at 0.05 IU/mL appeared to be optimal for the follicle culture in the domestic cats. Highlights • This is the first use of eCG supplementation in the in vitro follicular culture medium of cat species. • ECG supports the follicular growth and steroidogenic gene expression (STAR) from the multi-layered secondary follicle stage. • The concentration of 0.05 IU/mL eCG provides the best results. [ABSTRACT FROM AUTHOR]

AI was first reported in cats almost 50 years ago but, unlike AI in other domesticated animals (e.g. dogs, cattle, horses), has not been widely used for routine propagation by veterinarians or breeders. Anatomical and physiological challenges with cats have hindered the efficiency of AI using standardised transcervical approaches applied to other species. Development of laparoscopic oviductal AI (LO-AI) has helped overcome some of these barriers and, during the past 7 years, produced high pregnancy percentages (>70%) in domestic cats using both fresh collected and frozen–thawed semen and resulted in the birth of full-term offspring in three cat hereditary disease models and six wild cat species (ocelot, Pallas's cat, fishing cat, sand cat, tiger, clouded leopard). The standard approach involves exogenous gonadotrophin treatment (typically equine chorionic gonadotrophin followed by porcine LH) to induce ovarian follicular growth and ovulation, with laparoscopic visualisation of the oviductal ostium for direct intraluminal insemination with low numbers of spermatozoa. Similar ovarian synchronisation and insemination approaches have been used with wild felids, but frequently must be refined on a species-by-species basis. From a practical perspective, LO-AI in domestic cats now has adequate efficiency for applied use as a reproductive service in veterinary practices that possess basic laparoscopy expertise. [ABSTRACT FROM AUTHOR]

Abstract In the domestic cat, nuclear maturation and embryo development after vitrification of immature oocytes have been obtained but developmental competence after warming remains low. It has been reported that during folliculogenesis, the association and communication between the oocyte and the surrounding cumulus cells through connexin-based gap junctions is essential for normal oocyte and follicular development. Gap junctions result from the head-to-head interaction of two hemichannels; however, there is always a population of hemichannels not incorporated into gap junctions. These unopposed hemichannels are normally closed but may open under certain stress conditions, potentially also during vitrification and warming, turning them into toxic pores inducing cell injury and cell death. The aim of our study was to test whether inhibiting connexin 37 (Cx37) and connexin 43 (Cx43) channels with the connexin-targeting peptide Gap26 during vitrification and warming of cat immature cumulus-oocyte-complexes (COCs) could improve oocyte maturation and competence of resultant blastocysts derived by parthenogenetic activation. In the first experiment, our immunostainings confirmed the presence of Cx43 protein in the cytoplasm of immature cat oocytes and in the plasma membranes of cumulus cells. In the second experiment, COCs were randomly divided in three different groups: a control group (control), a group vitrified without Gap26 (vitrified) and a group vitrified with Gap26 (vitrified-peptide). The maturation rate was checked and oocytes from all three different experimental groups were parthenogenetically activated and cultured in vitro until day 8. After vitrification and warming, 49% of the oocytes in the control group matured, while this was 8% and 19% in the vitrified and vitrified-peptide groups, respectively. Compared to the vitrified group, oocytes in the vitrified-peptide group had significantly larger maturation rates. No blastocysts were detected at day 8 in the vitrified group, while 2% and 13% of the oocytes further developed to blastocyst at day 8 in the vitrified-peptide and control non-vitrified group, respectively. We conclude that the use of Gap26 in vitrification and warming media to vitrify immature cat oocytes improves maturation success and allows such oocytes to reach the blastocyst stage (2%) at day 8 after parthenogenetic activation. Highlights • Connexin 43 is present in immature cat cumulus-oocyte-complexes. • Maturation after vitrification of immature cat oocytes can be improved by Gap26. • Blastocysts are formed after vitrification of immature cat oocytes with Gap26. [ABSTRACT FROM AUTHOR]

Highlights • Toluidine blue staining is effective in the evaluation of feline sperm. • Head dimensions of feline spermatozoa in the different epididymal portions. • Feline spermatozoa with larger head presented looser chromatin packaging. Abstract When using assisted reproductive technologies, there is seldom an evaluation of DNA integrity during sperm analysis, which is an important variable for proper embryo development. The toluidine blue staining technique allows the simultaneous evaluation of sperm chromatin and sperm head dimensions. The objectives of this study were to evaluate the applicability of the toluidine blue staining method for analyzing DNA abnormalities in epididymal sperm (from the caput, corpus, and cauda) of cats and to investigate the correlations among DNA condensation, morphology, and sperm head dimensions. The DNA alteration indexes were obtained using the toluidine blue and acridine orange techniques, and comparisons of these indexes indicated there was a 65.4% (r = 0.654; P < 0.001) correlation. The sperm from the cauda had greater chromatin stability (97.9%) than the sperm from the epididymal head (92.1%; P = 0.0023). There, however, was no difference in chromatin stability between sperm obtained from the corpus and cauda regions, indicating that these sperm were already mature. The sperm head dimension was correlated with chromatin decondensation, and the sperm head size decreased as the sperm were transported through the three epididymal regions (P < 0.0001). In addition, the percentage of sperm that were deficient in chromatin condensation decreased as the sperm were transported through the epididymal caput, corpus and cauda (26.4, 15.7, and 3.4%, respectively; P < 0.0001). Thus, the sperm head size predicts the quality of chromatin condensation in sperm cells. [ABSTRACT FROM AUTHOR]

Kisspeptin is well known for its indispensable role in the regulation of reproduction, mainly through controlling the release of GnRH at the hypothalamic level. Recent studies have shown that kisspeptin and the kisspeptin receptor are expressed in the ovary and uterus, indicating an additional local function in reproduction at the extra-hypothalamic level. In this study, we aimed: (1) to investigate the localization pattern of kisspeptin and its receptor in the domestic cat ovary and uterus throughout the ovarian cycle using immunohistochemistry; and (2) to compare the relative expression of ovarian Kiss1 mRNA levels at different ovarian stages with qPCR analysis. Ovaries and uteri were collected and classified into three ovarian stages (inactive, follicular and luteal stages (n = 7 in each stage)) according to the ovarian morphology, vaginal cytology and serum progesterone. Kisspeptin immunoreactivity (Kp-IR) and kisspeptin receptor immunoreactivity (KpR-IR) were present in the ovaries and uteri at all ovarian stages, with no notable differences in the localization patterns between the ovarian stages. In the ovary, Kp-IR and KpR-IR were present in various ovarian compartments, including the follicles at all classes and the corpus luteum (CL). In the follicles, Kp-IR and KpR-IR were present in the oocytes, granulosa cells and theca cells. Kp-IR was also detected in the follicular fluid of antral follicles. In CL, a strong intensity of Kp-IR was present in the periphery CL of development/maintenance, with a relatively fainter intensity in the central CL. By contrast, KpR-IR was present in both peripheral and central CL at the same intensity. In the uterus, Kp-IR and KpR-IR were present in the uterine glands, myometrium and perimetrium. The relative ovarian Kiss1 mRNA level was higher in the follicular stage than in the luteal stage (P < 0.05). We concluded that kisspeptin and its receptor are present in the cat ovary and uterus, suggesting possible local functions of kisspeptin at the extra-hypothalamic level, such as folliculogenesis, oocyte survival and uterine adenogenesis. [ABSTRACT FROM AUTHOR]

The testicles are the primary sexual organs of male and their function is to produce sperm and sexual hormones. Disorders of the testicles are common in domestic cats. Therefore, detailed assessment of the testes is of great importance in veterinary medicine. Considering the recent advances in diagnostic imaging in companion animals, this review aims to describe the applicability elastography (qualitative and quantitative), Doppler, contrast-enhanced ultrasonography and B-mode ultrasonography in testes evaluation in cats. B-mode ultrasonography of the testicles combined with haemodynamic analysis in real time by Doppler and contrast enhanced ultrasonography can assist as diagnostic tool in evaluating testicular abnormalities in sick cats. Furthermore, ARFI elastography is a new ultrasound method that evaluates tissue elasticity by elastogram and shear weave. Ultrasonographic study of the testes is a common diagnostic imaging procedure. [ABSTRACT FROM AUTHOR]

The aim of the study was to determine the time after treatment with a 4.7 mg deslorelin implant until Tomcat spermatogenesis activity was restored, and seminal parameters reached pre-implant values. Tomcats (n = 6) were randomly assigned to one of two treatments. Three cats (n = 3) received a deslorelin implant (4.7 mG; Suprelorin ® , Virbac, France) in the interscapular subcutaneous region whereas three (n = 3) received no implant and served as control group. Semen samples were collected by electroejaculation every 4 wk from 3 mo before treatment (pretreatment samples) until reestablishment of pre-treatment sperm quality, 32 mo post-implant insertion (PI). Each semen sample was assessed for motility, velocity, concentration, total sperm count, viability, acrosome integrity, plasma membrane integrity and sperm morphology. After semen collection, testicular volume and presence/absence of penile spines were recorded. Additionally, blood samples were taken to measure testosterone concentration. An increase in sperm concentration and total sperm count was present 1 mo PI despite of an abrupt decrease in serum testosterone concentrations after 2–4 weeks. This initial stimulatory effect was followed by a decrease in seminal parameters, reduction of testicular volume and disappearance of penile spines 2 mo PI. A single Suprelorin ® 4.7 mg implant suppressed sperm production for 22–25 months. No clinically side effect was observed during the study period. All toms returned to their initial seminal quality 23–28 months after treatment. Therefore, we conclude that Suprelorin ® 4.7 mg is a safe option for reversible reproduction control during long periods in tomcats. [ABSTRACT FROM AUTHOR]

This study determined the efficacy of a combination of aglepristone and cabergoline on termination of mid-term cat pregnancies. Twenty cats with unwanted pregnancies between 30-40 days were included in the study. Aglepristone (10 mg/kg, sc) was given to the AGL group (n=6) twice in a 24-h interval. Cabergoline (5 μg/kg, peros) was administered to the CBG group (n=7) once daily until abortion started or for 8 days. AGL+CBG (n=7) received a combined treatment with both drugs. Abortion occurred in 50% of cats in the AGL group, 71.4% in the CBG group, and 100% in the AGL+CBG. However, the completion of pregnancy termination rate was 85.7% because of fetal retention in one cat from the AGL+CBG group. The interval between treatment-start of abortion (T-SA) was shorter in the AGL+CBG group (3.6±0.3 days) than in the AGL (6.5±0.0) and CBG (6.2±0.2) groups (P<0.01). Similarly, the interval between treatment-end of abortion (T-EA) was shorter in the combined group (4.3±0.5 days) than the others (7.3±0.3 and 6.9±0.9 days, respectively) (P<0.01). Decreasing in progesterone concentration was non-significant in the AGL group from the start of treatment to abortion completion day (dA/d8), but significant in the others (P<0.001). On dA/d8, it was significantly lower in the CBG group (P<0.01) and combined group (P<0.01) than in the AGL group. Only slight diarrhea was observed in 15.4% of the AGL+CBG group. In conclusion, the AGL+CBG combination increased the rate of abortion induction and significantly shortened T-SA and T-EA with negligible side effects. [ABSTRACT FROM AUTHOR]

We aimed to evaluate the effect of two vitrification methods on the morphology and functionality of vitrified feline preantral follicles. Feline ovarian tissue was vitrified with EG + trehalose combined or not with dimethyl sulphoxide (DMSO), using two different techniques (open or closed systems). Morphology, developmental capacity and mRNA expression of markers for follicle survival and quality were assessed before and after in vitro culture (IVC). Both vitrification and culture media were serum-free. Vitrification of feline ovarian tissue from five adult domestic cats was performed with EG + trehalose combined or not with DMSO. Two systems were used: the open system solid-surface vitrification (SSV) and the closed system ovarian tissue cryosystem (OTC). Histological analysis of follicle integrity showed that the percentages of normal follicles in previously vitrified ovarian fragments decreased after 7 days of in vitro culture (IVC), independently of the protocol used. Although follicular activation was observed by Ki-67 labelling, this was accompanied by extensive follicular degeneration as detected by a 3–4-fold decrease in follicular density. Remarkable follicle activation was observed in the ovarian tissue vitrified using OTC and subjected to IVC, probably due to a higher rate of degeneration of developing follicles. Even with such follicular loss, the results are promising for the combination of EG + DMSO + trehalose in a serum-free medium when applying the SSV method, with this approach resulting in the highest rates of normal developing follicles (19%) after 7 days IVC, together with granulosa cells proliferating at the same rate observed in fresh tissue. [ABSTRACT FROM AUTHOR]

The quality of cat epididymal sperm cryopreserved and stored by four methods was assessed. Epididymal sperm were suspended in Tris-glucose-citrate egg yolk extender, loaded in 0.25 mL straws and then cryopreserved. The samples in a standard protocol (LN) were cryopreserved and stored in liquid nitrogen (LN 2 ). The sperm straws in the LN-Fr-LN group were cryopreserved in LN 2 and stored in a −75 °C freezer; the straws were returned to LN 2 prior to thawing. The loaded straws in the Fr group were transferred directly from 4 °C to the freezer and maintained in the freezer until thawing. The Fr-LN samples were cryopreserved and stored in the freezer and were introduced into LN 2 before thawing. The sperm thawing was conducted on days 30, 60, 90 and 120 of cryopreservation. The sperm motility, viability, membrane integrity and acrosome integrity were evaluated at 15 and 180 min after thawing. The quality of post-thaw sperm in all three modified protocols was comparable (P > 0.05) and did not differ from that in the standard protocol except the membrane integrity of the 60 days stored samples evaluated at 15 min after thawing, which was significantly higher for the LN-Fr-LN than the Fr-LN groups (P = 0.04). The length of cryopreservation time had no effect (P > 0.05) on the sperm parameters assessed at 15 min after thawing. The sperm motility was significantly greater (P = 0.01 to P = 0.02) for the 15 min than the 180 min incubation. In conclusion, cat epididymal sperm could alternatively be cryopreserved and/or stored by using the −75 °C freezer for 120 days. To use, the cryopreserved sperm in the freezer could be thawed immediately or after being transferred to LN 2 . This was useful for the application of the −75 °C cryopreserved sperm in remote areas. [ABSTRACT FROM AUTHOR]

The reduced number of animals in most wild felid populations implies a loss of genetic diversity. The death of juveniles, prior to the production of mature sperm, represents a loss of potential genetic contribution to future populations. Since 2011 mouse testicular organ culture has introduced an alternative mechanism to produce sperm in vitro from immature tissue. However, extension of this technology to other species has remained limited. We have used the domestic cat (Felis catus) as a model for wild felids to investigate spermatogenesis initiation and regulation, with the mouse serving as a control species. Testicular tissue fragments were cultured in control medium or medium supplemented with knockout serum replacement (KSR), AlbuMax, beta-estradiol or AlbuMax plus beta-estradiol. Contrary to expectations, and unlike results obtained in mouse controls, no germ cell differentiation could be detected. The only germ cells observed after six weeks of culture were spermatogonia regardless of the initial stage of tubule development in the donor tissue. Moreover, the number of spermatogonia decreased with time in culture in all media tested, especially in the medium supplemented with KSR, while AlbuMax had a slight protective effect. The combination of AlbuMax and beta-estradiol led to an increase in the area occupied by seminiferous tubules, and thus to an increase in total number of spermatogonial cells. Considering all the media combinations tested the stimulus for felid germ cell differentiation in this type of system seems to be different from the mouse. Studies using other triggers of differentiation and tissue survival factors should be performed to pursue this technology for the genetic diversity preservation in wild felids. [ABSTRACT FROM AUTHOR]

The objective of this study was to identify and compare domestic feline sperm subpopulations, chilled at −1 °C for 24 and 48 h, as well as to analyze the sperm frequency in different subpopulations. Ten adult cats were used. Sperm collection was performed using electroejaculation (EEJ). Spermatic kinetics were evaluated using a computerized system at three moments: fresh, 24 and 48 h after refrigeration. The ejaculates were divided into a group refrigerated at −1 °C (n = 5,) and a group refrigerated at 4 °C (n = 5),. A total of 1560 spermatozoa were analyzed individually, and the sperm subpopulations were identified using multivariate statistics. Three spermatic subpopulations were defined using prior analysis of the hierarchical dendrogram. A principal components analysis (PCA) identified the existence of three groups with higher iterations at the three moments: PC1 (VAP, VCL, VSL, ALH, SVI), PC2 (STR, LIN, WOB and SMI) and PC3 (BCF). Subpopulation 1, after 48 h of refrigeration at −1 °C, and subpopulation 3, after 24 h of refrigeration at 4 °C, maintained their sperm quality, which allowed us to characterize the groups of spermatozoa that were resistant to cryopreservation. The present study identified three well defined ejaculate spermatozoa subpopulations, with proportional distributions between the groups and two refrigeration resistant subpopulations. [ABSTRACT FROM AUTHOR]

Contents: Cooling stored epididymal samples for several days allows facilities to transport and process genetic material post‐mortem. Improvements to this practice allow the preservation of sperm from domestic cats, which are the ideal study model for wild felids. However, the modifications in spermatic features and the oxidative profile are not fully understood in cats. This information is necessary for the development of biotechniques, such as new extenders for cryopreservation. Therefore, the purpose of this study was to evaluate the spermatic and oxidative profile in samples from the epididymal cauda of domestic cats cooled at 5°C for 24, 48 and 72 hr. Spermatozoa were collected from the epididymis cauda. Evaluations consisted of computer‐assisted sperm analysis (CASA), plasma membrane integrity (eosin/nigrosin), acrosome integrity (fast green/rose bengal), sperm morphology, sperm DNA integrity (toluidine blue), mitochondrial activity (3′3 diaminobenzidine), activity of the antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD), measurement of lipid peroxidation (TBARS) and protein oxidation. A decrease in sperm motility parameters was observed after 72 hr of cooling (i.e. total and progressive) with a higher percentage of minor (37.7 ± 6.3%) and total defects (53.4 ± 6.3%). Additionally, a decrease in high mitochondrial activity (Class I: 16.6 ± 2.2%) occurred after 72 hr. The decrease in motility rates after a long cooling time probably was caused by the increase in sperm abnormalities. A long cooling time causes cold shock and mitochondrial exhaustion, but there was no observed change with the oxidative stress condition. Therefore, cat epididymal sperm stored at 5°C appear to maintain a high quality for up to 48 hr of cooling time. [ABSTRACT FROM AUTHOR]

In cases where semen collection in tom-cats is not possible, FNA of testes is the alternative to evaluate sperm production. Although this technique for the diagnosis of fertility problems has been well discussed in other mammals (men, dogs, stallions), data for domestic cats are limited. Therefore, the aim of this study was to verify the reliability of FNA using needles of small diameters (22G and 29G) in testes of domestic cats of different ages to assess the spermatogenesis status and to present description of germ cells and Sertoli cells for cytological examinations. Thirty-four mixed breed cats aged between four months and two years presented for neutering to a Veterinary Hospital were used in this study. Under general anesthesia, testicular measures and FNA were followed by orchiectomy and imprints of the parenchyma of testes and epididymides. Cats were assigned into 3 groups: (G1) 10 cats aged less than 6 months, (G2) 14 cats aged between 6 months and one year and (G3) 10 cats aged more than one year. Cats weighted between 1.5 and 6.0 kg. The mean testicular volume (TV cm 3 ) was 0.55 (G1), 1.18 (G2) and 2.66 (G3). Hemorrhages in the needle path were observed in more than 70% of testes. Few samples (4/68) were excluded due to blood contamination. All germ cells and Sertoli cells were identified and quantified in imprint and FNA smears. Incomplete spermatogenesis was observed in cats aged less than 6 months using both techniques (FNA and imprint); therefore, testicular FNA should not be recommended for cats at this age. Complete spermatogenesis was found in 64% of cats aged from 6 months up to one year and in all cats aged more than one year. There were no differences of Sertoli cell Index (SEI) and Spermatic Index (SI) between FNA and imprints of cats older than 6 months. In conclusion, FNA using needles of small diameter in the testes of domestic cats is viable, reliable and can be used as a tool for the analysis of the spermatogenesis status of cats older than 6 months, mainly in cases in which semen collection is not possible. [ABSTRACT FROM AUTHOR]

Molar changes of the placenta are exceptionally rare in animals and in the cat, only one case of partial hydatidiform mole in a stillborn kitten was reported. A 3-year-old female cat was referred for anorexia, vomiting, depression and vaginal discharge. Abdominal distention and pain were noted. Blood count abnormalities were also observed in the cat. A septated, enlarged uterus with anechogenic content was diagnosed on ultrasonographic examination. Ventral midline ovariohysterectomy was performed. An uneventful recovery was observed and total blood count was within normal range on day 3 following ovariohysterectomy. Partial hydatidiform mole diagnosis was made based on the presence of embryos, gross appearance and histopathological findings. This report reflects the clinical presentation and histopathological findings of a partial hydatidiform mole leading to pregnancy loss in the cat. [ABSTRACT FROM AUTHOR]

Ovarian tissue transplantation could be a valuable technique for the preservation of endangered animals. The domestic cat affords an adequate experimental model for studies aimed at wild felids due to its phylogenetic similarity. Thus, this pilot study evaluated the efficacy of cat ovarian tissue autotransplantation to a peripheral site. Three adult queens were submitted to ovariohysterectomy. The ovaries were fragmented into eight pieces; two were fixed as a control and six were transplanted to subcutaneous tissue of the dorsal neck. Grafts were monitored weekly by ultrasound and fecal samples collected daily in order to monitor estradiol levels. Grafts were recovered on Days: 7, 14, 28, 49 and 63 post-transplantation for histological analyses. One graft was maintained in one animal for 8 months. A total of 2466 ovarian follicles were analyzed: 1406 primordial and 1060 growing follicles. All animals presented antral follicles in one or more of the grafts. The percentage of morphologically normal primordial follicles was always higher than 80%, except for Day 7 transplants. Although the proportion of growing follicles increased after transplantation, there was a general decrease in the percentage of morphologically normal growing follicles from Day 7 onwards. All animals demonstrated at least three estradiol peaks during the 63-day period, and one animal exhibited estrous behaviour on three occasions. Hormonal peaks directly correlated with the visualization of antral follicles (by ultrasound and/or histology) and the observation of estrous behaviour. Long-term results on one female showed the concentration of 37.8 pg/mL of serum estradiol on Day 233 post-grafting and the female exhibited estrous behaviour on several occasions. This graft showed one antral follicle, one luteinized follicle and two preantral follicles. In conclusion, cat ovary autotransplantation to the subcutaneous tissue restored ovarian function, with hormone production and antral follicle development, over both short and long term periods. This could be a valuable technique in the evaluation of ovarian cryopreservation methods in felids. Once the technique is shown successful, it may be applied in allografts or xenografts between different feline species. [ABSTRACT FROM AUTHOR]

The use of trap-neuter-return (TNR) as a method of managing free-roaming cat populations has increased in the United States in recent decades. Historically, TNR has been conducted most often at a grassroots level, which has led to inconsistent data collection and assessment practices. Consequently, a paucity of analyzable data exists. An initiative is underway to standardize TNR program data collection and assessment. However, it could be some time before scientifically sound protocols are implemented on a broad scale. In the interim, sets of data collected by nascent citizen scientists offer valid opportunities to evaluate grassroots TNR programs. The purpose of the present study was to examine the effectiveness of a TNR program conducted by a citizen scientist located in Chicago, Illinois, where a county law permitting TNR was enacted in 2007. Colony populations, when grouped by the number of years enrolled in the program, declined by a mean of 54% from entry and 82% from peak levels. Results from coexistent TNR programs in the Chicago area are consistent with these findings. [ABSTRACT FROM AUTHOR]

Approximately 50% of cats admitted to Australian shelters are kittens, and 26% of dogs are puppies, and, particularly for cats, euthanasia rates are often high. Cats can be pregnant by 4 months of age, yet the traditional desexing age is 5-6 months, and studies in Australasia and Nth America reveal that only a minority of veterinarians routinely perform early age desexing (EAD) of cats or dogs, suggesting they are not graduating with these skills. This study aimed to describe the attitudes of veterinary teaching staff in Australian and New Zealand universities towards EAD, and to determine if these changed from 2008 to 2015. It also aimed to identify students' practical exposure to EAD. Most (64%) of the 25 participants in 2015 did not advocate EAD in their teaching and, in their personal opinion, only 32% advocated it for cats. Concerns related to EAD cited by staff included anesthetic risk, orthopedic problems, hypoglycemia, and, in female dogs, urinary incontinence. Those who advocated EAD cited benefits of population control, ease of surgery and behavioral benefits. Only three of the eight universities provided a majority of students with an opportunity to gain exposure to EAD procedures before graduation, and in two of these, most students had an opportunity to perform EAD. In conclusion, most veterinary students in Australia and New Zealand are not graduating with the knowledge or skills to perform EAD, and have little opportunity while at university to gain practical exposure. Welfare agencies could partner with universities to enable students to experience EAD. [ABSTRACT FROM AUTHOR]

Freeze/thawing process reduces sperm survival and fertilizing ability of cat spermatozoa, with sperm motility being the most sensitive sperm parameter altered, due to cryo-damage. In this context, swim-up and density gradient processing methods can help to recover high motile and normal spermatozoa. Maximizing the use of frozen semen sample is essential, especially in endangered felids or high value cats in which sample size, number of samples or access to semen collection is reduced. To our knowledge, there is no previous report describing an in depth analysis of sperm motility improvement, after sperm selection techniques in frozen cat semen. Accordingly, we evaluated the effect of percoll gradient (PG) and swim up (SU) sperm selection techniques on sperm motility parameters and sperm recovery rate in frozen/thawed spermatozoa of domestic cat. Next, we evaluated the individual effect of the cat over sperm motility after PG sperm selection of frozen/thawed spermatozoa. SU and PG improved significantly all sperm motility parameters of frozen/thawed cat spermatozoa compared to simple washing. However, PG allows better sperm recovery from the original frozen sample and works mostly homogeneously among individual cats. This new information could help to maximize the use of frozen semen in endangered felids or high value domestic cats for its subsequent application on in vitro fertilization and artificial insemination. [ABSTRACT FROM AUTHOR]

Contents The objective of this study was to evaluate mitochondria in immature and in vitro-matured domestic cat oocytes and to assess for the first time the effect of vitrification on mitochondrial traits. Mitochondrial distribution and aggregation were assessed using confocal microscopy after staining with the fluorescent dye-MitoTracker® Red CMXRos. Only cells at the germinal vesicle and the metaphase II stages of nuclear development, representing immature and mature oocytes, respectively, were included in our study. Our study shows that 80% of immature and 100% of mature oocytes exhibit a peripheral pattern of mitochondria distribution, indicating that, in contrast to the situation in other species, the mitochondria of cat oocytes are not dispersed throughout the cell after in vitro maturation but instead maintain a strong affinity for the oocyte periphery near the membrane. However, a loss of aggregation was observed during in vitro maturation-78% of immature oocytes showed homogeneous granulation versus only 18% of mature oocytes ( p < .001). The increased intensity of MitoTracker® Red CMXRos staining after in vitro maturation ( p < .05) may be tentatively attributed to an increase in mitochondrial activity but could likewise reflect a concomitant appearance of sulphhydryl groups in cytoplasm (known to be targeted by the dye). Mitochondrial distribution did not change upon vitrification; however, dye intensity decreased ( p < .05) and mitochondrial aggregation was intensified in both immature and mature vitrified cat oocytes. [ABSTRACT FROM AUTHOR]

Contents This study is the first description of the distribution of mast cells in various phases of the oestrous cycle in the ovary of cat. Furthermore, this is the first description in species with an induced ovulation. The aim was to describe the distribution of mast cells and variability of their numbers in the feline ovaries in different phases of the oestrous cycle. The number of mast cells in medulla ovarii was affected by the estradiol and progesterone level in the blood serum because the lowest number was detected in anoestrus when the levels of hormones were basal. Nevertheless, both high and low numbers of mast cells were found in oestrus and dioestrus. To conclude, mast cells seem to be essential for the induction of spontaneous ovulation, but they do not play the same role for ovulation itself in cats with induced ovulation. [ABSTRACT FROM AUTHOR]

Contents In cats, accuracy of parturition day prediction by ultrasonographic measurement of foetal structures is decreasing towards the end of gestation. Foetal measurements during the last days of pregnancy are scarce. We determined foetal biparietal, abdominal and eye diameter ( BPD, AD and ED, respectively) by ultrasonography as well as maternal blood progesterone (P4) within five days of delivery to predict parturition date and calculate accuracy of prediction. Foetal BPD at birth was compared with newborn kitten head diameter ( HD). Kitten HD, crown-rump length ( CRL) and body weight were compared by breed and gender. Ultrasonography measurements were carried out on the day of parturition in 14 queens, and on days 62-63 after the first mating and repeated 24-72 hr later in ten other cats. Accuracy of parturition day prediction using BPD and AD was determined based on the equations of Beccaglia et al. (2008) Veterinary Research Communications, 32(Suppl 1), S99 and Garcia Mitacek et al. (2015) Theriogenology, 84, 1131. Progesterone was measured at the time of presentation and repeated 24-72 hr later if parturition did not occur. Data were analysed with linear regression, t test, Mann-Whitney U test, one-way anova and Kruskal-Wallis test. There was a moderate relationship between BPD, days before birth ( DBB) and litter size. AD and DBB had a low agreement, and ED was not associated with DBB. BPD at birth was significantly related to HD. The accuracy of parturition day prediction using BPD and AD was 27-53% and 17-35%, respectively. Kitten HD was associated with body weight, and both were inversely related to litter size. Newborn biometric measurements differed by breed but not by gender. Progesterone decreased towards parturition and reached 3.18 ± 1.68 ng/ml on the day of delivery. In conclusion, close to birth, the combination of foetal ultrasonography and maternal blood P4 rather than each as a sole predictor of parturition is recommended. [ABSTRACT FROM AUTHOR]

Contents Mitochondria play fundamental roles during oocyte development. The accumulation and spatial redistribution of these energy-producing organelles have been linked to the developmental competence of mammalian oocytes. Here, we assessed the copy number, distribution and activity of mitochondria within cat oocytes during folliculogenesis. In Experiment 1, oocytes were recovered from primordial ( n = 152), primary (112), secondary (95), early (131), small (118), antral (86) and advanced antral (5) stages follicles, and mitochondria DNA extracted and quantified using qPCR. In Experiment 2, oocytes from pre-antral ( n = 44), early antral ( n = 66), small antral ( n = 59), antral ( n = 41) and advanced antral ( n = 21) follicles were isolated and stained with CMXRos MitoTracker dye to assess mitochondrial distribution pattern and activity levels. Oocyte's mitochondria DNA (mt DNA) copy numbers gradually increased as folliculogenesis progressed, with a significant shift at the small antral stage (0.5 to <1 mm in diameter). The location of mitochondria gradually shifted from a homogeneous distribution throughout the cytoplasm in pre-antral oocytes to a pericortical concentration in the advanced antral stage. Quantification of CMXRos fluorescent intensity revealed a progressive increase in mitochondrial activity in oocytes from the pre-antral to the large antral follicles. Taken together, these findings demonstrated that cat oocytes undergo dynamic changes in mitochondrial copy number, distribution and activity during folliculogenesis. These significant modifications to this crucial cytoplasmic organelle are likely associated with the acquisition of developmental competency by cat oocytes. [ABSTRACT FROM AUTHOR]

Contents Sterilization is a key strategy to reduce the number of domestic cats entering and killed in shelters each year. However, surgical sterilization is expensive and labour-intensive and cannot fully address the 70 million free-roaming cats estimated to exist in the United States. GonaCon™ is a gonadotropin-releasing hormone vaccine originally developed for use as a wildlife immunocontraceptive. An earlier formulation was tested in domestic cats and found to be safe and effective for long-term contraception. However, the current Environmental Protection Agency ( EPA)-registered formulation consists of a different antigen-carrier protein and increased antigen concentration and has never been tested in cats. A pilot study was undertaken to evaluate the short-term safety of a single GonaCon immunization, assess the consequences of vaccinated cats receiving an accidental second GonaCon injection and determine the humoral immune response to immunization. During Phase 1, cats in Group A ( n = 3) received a single intramuscular injection of GonaCon and Group B ( n = 3) received a single intramuscular injection of saline. During Phase 2, Group A received a second GonaCon injection and Group B received their initial GonaCon injection. All cats developed Gn RH antibodies within 30 days of vaccine administration. The endpoint titre (1:1,024,000) was similar among all cats, and levels remained high throughout the duration of the study. Four cats developed a sterile, painless, self-limiting mass at the site of injection. The mean number of days to mass development was 110.3 (range, 18-249 days). In conclusion, this preliminary study suggests that the EPA-registered GonaCon formulation is safe for continued testing in domestic cats, an accidental revaccination should not increase the risk of a vaccine reaction and the EPA-registered formulation effectively elicits a strong humoral immune response. [ABSTRACT FROM AUTHOR]

Contents The objective of the study was to assess the efficacy of coculture with conspecific cumulus-denuded oocytes ( CDOs) during in vitro maturation in a three-dimensional system of barium alginate microcapsules on the in vitro embryo development of domestic cat cumulus-oocyte complexes ( COCs). In Experiment I, COCs were cocultured with conspecific CDOs or cultured separately in a 3D system for 24 hr of in vitro maturation, before assessing the meiotic progression. In Experiment II, the in vitro fertilization of COCs and CDOs was carried out with chilled epididymal spermatozoa and the presumptive zygotes were cultured in vitro separately for 7 days in 3D microcapsules before assesment of embryonic development. The results showed that the viability was maintained and that meiosis was resumed in the 3D culture system. The presence of CDOs during in vitro maturation improved the meiotic competence of the COCs, since the proportions of telophase I/metaphase II were higher than that in the groups cultured separately. The enrichment of the maturation system by companion oocytes also enhanced the ability of COCs to develop into embryos, and increased the percentages of morula and blastoycst stages. The COCs cocultured with CDOs developed at higher rates than the COCs cultured separately and the CDOs themselves. The beneficial effects of coculture with conspecific CDOs were presumably due to the paracrine action of some secreted factors that enhanced many molecular patterns related to the complex of cumulus oophorous cells. Further investigations to understand how the 3D microenvironment can influence the features of oocytes and embryos are required. [ABSTRACT FROM AUTHOR]

Contents Embryo vitrification is a modern technique for cryopreservation in assisted reproductive programs. From all the embryos, blastocysts are the most challenging during cryostorage due to their size, multicellular structure and the presence of blastocoelic fluid. The aim of this study was to evaluate the suitability for vitrification of various developmental stages of feline embryos and the influence of the artificial shrinkage ( AS) of expanded blastocyst on post-vitrification survival rates. The AS procedure is the manual puncture of the trophectoderm allowing for the reduction of blastocoelic fluid prior to vitrification and thus preventing the ice crystal formation. The vitrified embryos were divided into groups of 2-cell, 4- to 8-cell, >8-cell, morulae, compacted and expanded blastocyst, based on morphological assessment and vitrified in groups of 1-3 embryos per Cryotop. The post-warming survival was similar regardless the embryo developmental stage prior vitrification; however, development to blastocysts was only noted in 4- to 8-cell and >8-cell vitrified embryos (13% and 27%, respectively). Following warming, the significantly more viable blastocysts were noted in vitrified compacted versus expanded blastocyst and in expanded blastocyst subjected to AS procedure versus expanded blastocyst without AS (total survival: 58.3% vs. 33.3% and 64.3% vs. 38.5%, respectively; re-expansion rate within 2 hr post-warming: 41.7 vs. 6.7% and 50% vs. 7.7%, respectively). One-fifth of vitrified expanded blastocyst showed morphological damage immediately after warming procedure, whereas no visible damage was noted in compacted blastocyst and artificially collapsed expanded ones. The obtained results suggest that the most suitable for vitrification are feline embryos containing four to 16 blastomeres and compacted blastocyst. In addition, the reduction of blastocoel cavity in expanding blastocyst by artificial collapse improved the blastocyst vitrification outcome. [ABSTRACT FROM AUTHOR]

Contents During the last decade, analysis of anti-Müllerian hormone ( AMH), highly conserved between mammalian species, has contributed to new information in reproductive endocrinology, due to clinically available diagnostic assays. AMH is produced solely in the gonads, in the Sertoli cells of testes and granulosa cells of the ovary, and thus offers possibilities to diagnose physiologic and pathologic conditions involving these organs. This article reviews indications for AMH analysis in cats and dogs, including diagnosing the presence of gonads, and granulosa or Sertoli cell tumours. Diagnostic challenges are addressed. One specific organ, the prostate, is commonly affected by pathologic changes in older dogs. A commercial assay for analysing canine prostatic specific esterase ( CPSE) enables analysis of CPSE in clinical practice, of potential value in the workup of benign prostatic hyperplasia in male dogs. This is described in this review, as is a new method for analysis of steroids: liquid chromatography-tandem mass spectrometry LC- MS/ MS. Steroids have since long been analysed in studies on reproduction, and LC- MS/ MS has the advantage of allowing analysis of panels of multiple steroids from small sample volumes. Altogether, these available methods may give new insights into small animal reproduction and are valuable tools for the practicing veterinarian. [ABSTRACT FROM AUTHOR]

Contents This study aimed to evaluate and compare the ovarian and uterine characteristics along with the ovarian mRNA and protein expression of LHR and FSHR between the pre-pubertal and adult female cats. The uterine horns and ovaries were collected from pre-pubertal and adult female cats at their follicular, luteal and interoestrous stages of the oestrous cycle ( n = 6/group). Endometrial and myometrial thickness, uterine gland diameter, ovarian weight and type of follicles were analysed. The mRNA and protein expression of LHR and FSHR was analysed by IHC and qPCR, respectively. The ovarian weight of pre-pubertal cats was significantly lower than that of adult cats. No differences were recorded in the numbers of primordial and primary follicles between the study groups, while adult luteal cats had significantly lower numbers of antral follicles compared to pre-pubertal cats. No differences in the ovarian expression of FSHR mRNA, LHR protein or mRNA were found between the pre-pubertal and adult cats, but significantly lower FSHR protein expression was found in pre-pubertal cats compared to adult luteal cats. [ABSTRACT FROM AUTHOR]

Contents Maturation of oocytes is a prerequisite for successful embryo development. The fertilization competence of in vivo derived oocytes is significantly higher than that of oocytes matured in vitro. Commonly evaluated morphological criteria for oocyte maturation do not reflect the complexity and quality of maturation processes. Oocytes and granulosa cells are communicating closely in a bidirectional way during follicular growth and maturation. Assessing the mRNA expression of specific genes in granulosa cells could be a non-invasive way to evaluate the conditions of in vitro oocyte maturation. The objective of this study was to elucidate the influence of two different FSH additives on the in vitro maturation rate and gene expression of cumulus-oocytes complexes in domestic cat. Feline oocytes were matured in a medium, supplemented with LH and 0.02 IU/ml porcine FSH versus 0.02 IU or 1.06 IU/ml human FSH. Granulosa cells were separated from oocytes directly after 24 hr of maturation or after additional 12 hr of in vitro fertilization. Gene expression levels were analysed by quantitative PCR for aromatase, antimullerian hormone, follicle stimulating hormone receptor ( FSHR), luteinizing hormone/choriogonadotropin receptor ( LHCGR) and prostaglandin E synthase. Neither oocyte maturation rate nor gene expression levels differed after 24 or 36 hr in all three groups. However, variations were discovered in correlations of expression levels, for instance for FSHR and LHCG, indicating differences in the fine-tuning of in vitro maturation processes under varying FSH supplementations. We suppose that correlation between gene expressions of selected genes suggests a superior maturation quality of feline oocytes. [ABSTRACT FROM AUTHOR]

Contents Up to date, in vitro maturation (IVM) rates of oocytes are highly variable between individual cats. This study was carried out to investigate the predictive value of age and anti-Müllerian hormone (AMH) concentration in relation to capacity for IVM of cat oocytes. Ovaries were collected from 33 cats, which were divided into three age groups: (i) 0-3 months (pre-pubertal); (ii) 3-12 months (peripubertal); and (iii) older than 12 months (pubertal). The cumulus-oocyte complexes (COCs) were matured and subsequently stained to check nuclear maturation status, and blood was taken for AMH analysis. Increasing age was significantly associated with decreasing AMH levels, and mean AMH levels differed significantly between all age categories: group 1: mean AMH 18.71 μg/L; group 2: mean AMH 9.27 μg/L; and group 3: mean AMH 4.13 μg/L. Moreover, the probability of maturation was more likely in groups 2 and 3 compared to group 1. Between categories 2 and 3, no significant difference in maturation probability was found ( p = .31). Finally, the probability of oocyte maturation decreased significantly with increasing AMH levels. In age group 2, oocytes with a higher AMH level were less likely to mature. In age groups 1 and 3, no significant association between the AMH level and the proportion of maturated COC was found. We can conclude that if a higher probability of nuclear maturation is required, it is preferable to use cats with lower AMH levels and older than 3 months of age to improve cat IVM. [ABSTRACT FROM AUTHOR]

Contents The aim of this study was to evaluate the effect of co-culture of denuded oocytes with cumulus cells ( CC) or cumulus-oocyte complexes ( COCs) on in vitro maturation ( IVM) and in vitro fertilization ( IVF). Immature oocytes were collected from ovaries of domestic cats following a routine ovariectomy. Oocytes were matured in vitro for 24 hr within four groups: (i) denuded oocytes ( DO), (ii) DO co-cultured with CC, (iii) DO co-cultured with COC and (iv) COC as a control group. In further experiments, COCs were matured in vitro for 24 hr, and then, oocytes were randomly divided into four groups as previously described and fertilized in vitro. Embryos were cultured for up to 7 days. At the end of each experiment, oocytes/embryos were stained with Hoechst 33342 solution and observed under an inverted fluorescence microscope. The results of oocyte maturation showed that their meiotic competence decreased significantly in all experimental groups, compared to the control group. The maturation rates were approximately 45%, 24%, 43% and 76% in experiment 1, and 21%, 14%, 33% and 50% in experiment 2 in groups (i), (ii), (iii) and (iv), respectively. Examination of in vitro fertilization revealed that embryos developed up to the morula stage in all experimental groups. DO and oocytes cultured with COC during fertilization showed a lower cleavage rate-36% and 25% as opposed to those co-cultured with loose CC and the control group-43% and 42%, respectively. Results of this study indicate that cumulus cells connected with an oocyte into a cumulus-oocyte complex are irreplaceable for the maturation of domestic cat oocyte, but that the addition of loose CC may be beneficial for IVF. [ABSTRACT FROM AUTHOR]

Contents Reproduction management and performances are evaluated in the feline species only through a limited number of animals and studies. Our objective was to provide reference figures in purebred cats, from a large-scale sample. Data were collected from an online software dedicated to cattery management (Breeding Management System®, BMS, Royal Canin, Aimargues, France). Information was recorded on a voluntary basis by French breeders between 2011 and 2014. Data were anonymously transferred for analysis. A total of 9,063 oestrous periods (in contact with a male) from 5,303 queens (45 breeds) were recorded from 1,521 breeders. Most matings (70.1%) occurred during increasing day length periods. The mean age at mating (± SD) was 2.7 ± 1.6 years for queens and 2.9 ± 1.9 years for tomcats. Pregnancy rate (based on breeders declaration) was 85.2%. Among queens declared pregnant, 8.4% failed to maintain pregnancy. Globally, 78% of the mated females gave birth to 28,065 kittens within 7,075 L. Mean litter size was 4.0 ± 1.9 kittens among which 8.5% were stillborn. Neonatal and paediatric mortality rate was 8.2%. In total, 16.0% of kittens born died before weaning. The results of this study are based on the largest feline database ever analysed. The figures collected can thus be used as reference to define average reproductive performances in numerous breeds for cat breeders. Further analysis will identify factors influencing reproductive performances and early mortality in the feline species. [ABSTRACT FROM AUTHOR]

Contents Foetal gonads already produce steroid hormones and by this influence the further development of external and internal genitalia as well as of the brain. Beside this, foetal gonads themselves can be influenced by foetal or maternal hormones. The time course of foetal gonadal development can differ between species. As knowledge on processes in domestic cats is very limited, the steroidogenic enzyme expressions as well as these of steroid receptors were analysed in foetal gonads of domestic cats. We investigated a period from beginning of the second half of pregnancy to the beginning of the third trimester; a phase, where also gonadal development proceeds. The mRNA expression of most of the steroidogenic enzymes was remarkably higher in male gonads compared to female ones on all analysed days. The enzyme mRNA expression in female gonads shows a tendency for an increase towards the beginning of the third trimester, except that of aromatase gene CYP19A1-it shows the opposite trend. CYP19A1 was detectable just in female gonads, indicating that only female foetal gonads are capable of producing oestrogens. Gene expressions of genomically and non-genomically acting steroid receptors for progesterone, androgen and oestrogen reception were observed in gonads of both genders. Slightly higher expressions of some receptors were detected in female compared to male gonads; only for the non-genomically oestrogen receptor GPER, we observed the opposite. The protein staining for progesterone receptor membrane component 1 ( PGRMC1) exposed a potential function of it on steroid-producing cell and/or cells that suppose early oogenesis. [ABSTRACT FROM AUTHOR]

Contents Measurement of circulating luteinizing hormone ( LH) concentrations in cats and temporal changes following ovariohysterectomy ( OHE) or possibly Gn RH vaccination may be informative for assessing their fertility, contraception or sterilization status. In this study, serum LH concentrations were measured in domestic cats ( n = 6) immediately prior to and up to 120 days post- OHE. Basal LH concentrations of females previously subjected to OHE ( n = 4; ~1.5 years post- OHE) were compared pre- and post-vaccination with a Gn RH immunocontraceptive, and to LH concentrations in intact females. Basal serum LH concentrations (2.67 ± 0.43 ng/ml; mean ± SEM) in intact females increased ( p < .01) by 30 days post- OHE (5.65 ± 0.87 ng/ml) but then declined ( p < .05) to pre- OHE levels (mean range, 3.26-3.62 ng/ml) at days 60-120 post- OHE. Serum LH (3.84 ± 0.51 ng/ml) in four females ~1.5 years after OHE tended to be higher ( p = .10) than those of intact females prior to OHE. Three months following first or second Gn RH immunocontraceptive vaccine treatment, serum LH values in females previously subjected to OHE decreased ( p < .05) to concentrations similar to those observed in intact females. Our preliminary results suggest that OHE of domestic cats causes a marked increase in basal LH levels within the first few weeks after ovariohysterectomy followed by a return to pre- OHE basal values over the next several months. Reduced LH concentrations after Gn RH vaccine may indicate the effectiveness of the immunocontraceptive in reducing the circulating levels of Gn RH, thereby reducing secretion of LH. [ABSTRACT FROM AUTHOR]

Contents Ovarian tissue cryopreservation followed by tissue culture is a promising approach to preserving the fertility of biomedical models and endangered species. The objective of this study was to investigate the impact of exposure time to vitrification solution and presence of sucrose using different exposure temperatures and base media on intra-ovarian follicle integrity. Peripubertal ovarian cortical pieces were obtained by isolating the cortex and dissecting it into 1 × 1 × 0.2 mm3 pieces. The cortical pieces were then exposed to equilibration solution and then vitrification solutions ( VS) in one of the conditions mentioned above, plunged directly into liquid nitrogen and stored for ≥24 hr in liquid nitrogen. After thawing, the cortical pieces were cultured in vitro for 0, 1 or 7 days to determine the follicle integrity (through histological assessment) and the ability of the tissue to recover from cryoinjury. Fresh controls maintained a constant level of normal morphology (>60% of the total follicles) throughout the culture period. Cortical pieces exposed to VS with sucrose for 10 min had the highest percentage of normal follicles (approximately 20% after 7 days of culture) throughout the culture period. Other conditions using different base medium, lower exposure temperatures or different thawing methods did not improve the follicle integrity. This protocol provides a solid foundation on which to optimize ovarian tissue cryopreservation in the domestic cat and to investigate the molecular effects of vitrification. [ABSTRACT FROM AUTHOR]

Contents Spermatogonial stem cells ( SSCs) are an important tool for fertility preservation and species conservation. The ability to expand SSCs by in vitro culture is a crucial premise for their use in assisted reproduction. Because SSCs represent a small proportion of the germ cells in the adult testis, culture success is aided by pre-enrichment through sorting techniques based on cell surface-specific markers. Given the importance of the domestic cat as a model for conservation of endangered wild felids, herein we sought to examine culture conditions as well as molecular markers for cat SSCs. Using a cell culture medium for mouse SSCs supplemented with glial cell-derived neurotrophic factor ( GDNF), germ cells from prepuberal cat testes remained viable in culture for up to 43 days. Immunohistochemistry for promyelocytic leukaemia zinc finger ( PLZF) protein on foetal, prepuberal and adult testis sections revealed a pattern of expression consistent with the labelling of undifferentiated spermatogonia. Fluorescence-activated cell sorting ( FACS) with an antibody against epithelial cell adhesion molecule ( EPCAM) was used to sort live cells. Then, the gene expression profile of EPCAM-sorted cells was investigated through RT- qPCR. Notably, EPCAM (+) cells expressed relatively high levels of CKIT ( CD117), a surface protein typically expressed in differentiating germ cells but not SSCs. Conversely, EPCAM (-) cells expressed relatively high levels of POU domain class 5 transcription factor 1 ( POU1F5 or OCT4), clearly a germ line stem cell marker. These results suggest that cat SSCs would probably be found within the population of EPCAM (-) cells. Future studies should identify additional surface markers that alone or in combination can be used to further enrich SSCs from cat germ cells. [ABSTRACT FROM AUTHOR]

Contents The cryopreservation of testicular tissue is presented as the only alternative for the preservation of genetic material from prepubertal animals. However, this biotechnology is still being tested. The objective of this study was to evaluate the effect of different associations of cryoprotectants and the potential of cell proliferation after vitrification of testicular tissue of prepubertal cats. Five testicular pairs from five prepubertal cats were used, and each pair was divided into four fragments. Of these, one fragment composed of the control group ( CG) and the rest were distributed in experimental groups according to the associations of cryoprotectants to be tested (dimethyl sulphoxide ( DMSO)/glycerol ( GLY); ethylene glycol ( EG)/ GLY) or DMSO/ EG) in a final cryoprotectant concentration of 5.6 m. The fragments were submitted to vitrification, and after one week, fragments were heated and processed for histomorphological evaluation and quantification of nucleolar organizer regions ( NORs). DMSO/ GLY did not differ from CG and was superior to the other vitrified groups, as to cell separation and degree of shrinkage of the basal membrane. Concerning cell differentiation, visibility of the nucleus and nuclear condensation, all the vitrified groups were inferior to CG; however, DMSO/ EG was inferior to DMSO/ GLY and EG/ GLY, which did not differ among themselves. CG was superior to all groups in quantification of NORs. DMSO/ EG was inferior to all others, and there was no difference between DMSO/ GLY and EG/ GLY. The association DMSO/ GLY presented the best preservation of tissue integrity and potential of cell proliferation after vitrification of the testicular tissue of prepubertal cats. [ABSTRACT FROM AUTHOR]

Contents Puppy and kitten survival over the first weeks is particularly dependent on colostrum, a specific secretion of the mammary gland produced during the first 2 days post-partum. Colostrum is a source of nutrients and immunoglobulins. It also contributes to the digestive tract maturation. Colostrum differentiates from milk mainly based on its concentration in immunoglobulins G: 20-30 g/L in dog colostrum, 40-50 g/L in cats' vs <1 g/L in milk. IgG concentration rapidly drops after parturition (−50% in 24 hr). Immune quality of colostrum is highly variable between bitches, with no relationship with maternal blood IgG level, dam's age, breed size or litter size. In addition to systemic immune protection, colostrum also plays a major role for local digestive protection, due to IgA, lysozyme, lactoferrin, white blood cells and various cytokines. Energetic concentration of canine and feline colostrum is not superior to that of mature milk. It depends on colostrum fat concentration and is affected by breed size (higher in breeds <10 kg adult body weight). As puppies and kittens are almost agammaglobulinemic at birth, transfer of IgG from their digestive tract into their bloodstream is crucial for their survival, IgG absorption ending at 12-16 hr after birth. Energetic supply over the two first days of life, as evidenced by growth rate over the two first days of life, also affects risk of neonatal mortality. Early and sufficient suckling of colostrum is thus the very first care to be provided to newborns for their later health and survival. [ABSTRACT FROM AUTHOR]

Contents The sperm centrosome is an essential organelle with a key role in organizing the sperm aster for proper syngamy and formation of the first mitotic spindle. The sperm cell acquires the functional capability during epididymal transit by incorporation of key factors. The objective of the study was to identify these key maturation proteins, such as ninein and centriolin as well as cenexin-a scaffold protein that serves to bind ninein and centriolin. Epididymal samples were dissected from 17 adult cat testes (>1 year old) and spermatozoa were extracted from the different regions, including rete testis, caput, corpus, cauda and vas deferens. Tissue samples and sperm cells were fixed separately in 4% paraformaldehyde before immunostaining with anticenexin, ninein or centriolin antibodies. Results showed that the proportion of sperm cells with cenexin localized at the centrosome progressively increased along the tract with the lowest percentage of stained cells in the testis (mean = 45%) and highest in the cauda (mean = 81%). Although not significant, the intensity of cenexin immunofluorescence in positive cells increased twofold from the testis to vas deferens. There was no significant difference in the proportion of sperm labelled with centriolin or ninein (ranges of 21%-26% and 33%-48% between segments, respectively) or the intensity (±58% and ±63% change as compared to testis, respectively). Cenexin may serve as a scaffold protein for centriolin and ninein, as the vast majority of spermatozoa only displayed colocalization of these proteins when cenexin was also present (mean = 85% and 91% colocalization, respectively). In summary, these results could be applied to future efforts to create an in vitro culture system capable of rescuing the impaired centrosome of an infertile male, with particular potential for wild felid conservation. [ABSTRACT FROM AUTHOR]

Contents Assisted reproductive techniques are a valuable tool for conservation breeding of endangered species. Cryopreservation methods are the basis of gamete banks, supporting genetic diversity preservation. Unfortunately, cryopreservation of feline oocytes is still considered an experimental technique. The aim of this study was to compare two commercial kits, with our protocol for vitrification of cat oocytes ( IZW), which comprises a three-step method with ethylene glycol, DMSO, fetal calf serum, trehalose and Ficoll PM-70. Furthermore, we applied slush nitrogen ( SN2) for ultra-rapid freezing to improve survival rates. Cumulus-oocyte complexes were collected from domestic cat ovaries by slicing and vitrified at immature stage using Cryotop as storage device. Vit Kit® Freeze/Thaw ( n = 89) showed the lowest maturation percentage obtained after warming (10.1%). A significant difference in maturation percentage of oocytes was found between Kitazato® kit (38.7%, n = 137) and IZW protocol (24.5%, n = 143). The cleavage after ICSI of warmed and matured oocytes (20.7% and 28.6%, respectively) and the morula percentage (18. 2% and 22.5%, respectively), however, did not reveal any significant difference between the two methods. Application of SN2 did not result in any improvement of oocytes' cryopreservation. Maturation percentage of the oocytes vitrified by IZW method with SN2 ( n = 144) decreased until 6.1%, without any cleavage after fertilization. For Kitazato® ( n = 62), only 17.7% were able to undergo maturation and cleavage percentage dropped to 18.2%, not reaching morula stage. These data demonstrate that feline oocytes can be vitrified either by our IZW method or by commercial Kitazato® kit, but the use of SN2 is improving neither maturation nor cleavage percentages when combined with these procedures. [ABSTRACT FROM AUTHOR]

Contents In vitro culture of ovarian preantral follicles has emerged as a reproductive technology aimed at obtaining large amount of oocytes for in vitro embryo production. The addition of growth factors ( GF) in the in vitro culture of preantral follicles of different species has provided superior results of follicular development, antrum formation and proliferation of granulosa cells. However, there are only few reports regarding the use of these factors on feline preantral follicle in vitro culture. Thus, the aim of this study was to investigate the effect of a combination of IGF-1 and EGF on in vitro viability and growth of preantral follicles and enclosed oocytes collected from domestic cats. A total of 64 follicles characterized by multilayer granulosa cells were isolated and individually cultured for 6 days (T6) in minimum essential medium supplemented with IGF-1+ EGF (100 ng/ml each) or without (control). A higher percentage of follicles were viable after culture with GF than without, and an increase in size when IGF-1+ EGF were added to the medium (170 ± 32.4 μm (T0) vs. 201 ± 22.3 μm (T6); p < .05) was observed. An increase in the diameter was also observed in follicles cultured without GF, but this increase was only 8.3% compared to 15.4% of those cultured with GF ( p < .05). No differences were found in the diameter of oocytes contained in follicles cultured in the non-supplemented or supplemented media (107.9 ± 11.8 μm (T0) vs. 113.2 ± 15.6 μm (T6); p > .05). These data suggest that the addition of IGF-1 and EGF to the culture medium promotes the in vitro development of preantral follicles of cats. [ABSTRACT FROM AUTHOR]

Contents The gonadotropin-releasing hormone (Gn RH) stimulation test is a common procedure used to investigate normality of the pituitary-gonadal axis in mammals. There is very little information on the technique, its efficacy and side effects in small animals and in particular no information for male cats. In dogs, such test is performed by intravenous ( IV) administration. With cats, the number of times the animal needs to be restrained for blood sampling should be the least possible. The purpose of this study was to assess efficacy and side effects of the Gn RH stimulation test in tomcats comparing the IV with the intramuscular ( IM) route of administration. A Gn RH stimulation test was performed in eight adult tomcats through IM or IV administration of 50 μg gonadorelin. The response of the pituitary-gonadal axis was assessed by measuring serum testosterone on blood samples collected prior to and 1 hr following treatment. When considering each single group of cats, the post-stimulation serum testosterone values were significantly higher than the pre-treatment ones ( p < .05). When comparing the two groups of cats, basal testosterone concentrations did not differ, and also post-Gn RH testosterone concentrations did not differ. In conclusion, in the cats of our study, the Gn RH stimulation test produced the same results following the IM or the IV route of administration. Therefore, in tomcats, the IM route can be considered as effective as the IV one and should be preferred when doing a Gn RH test. [ABSTRACT FROM AUTHOR]

The aim of this study was to describe resistance index (RI) and systolic/diastolic ratio (S/D ratio) of uterine and umbilical arteries in an experimental model of abnormal pregnancy in felids. On days 30 to 35 (32 ± 2.9) after mating, 20 domestic short-hair pregnant queens were randomly assigned to one of the following treatment groups: a treated group (TG; n = 8), which received 10 mg/kg of aglepristone subcutaneously twice, 24 hours apart, and a control nontreated group (CG; n = 12). M-mode and Doppler ultrasonographic evaluations were performed at the initiation of the treatment (Day 0) and then every other day during 8 days. In both groups, uterine and umbilical arteries were evaluated by Doppler ultrasound, whereas fetal heart rate was assessed by M-mode ultrasound. Resistance index of uterine artery augmented in TG from Day 2 onward, conversely it decreased in CG (P < 0.01). On Day 8, RI values were 0.64 ± 0.05 vs 0.37 ± 0.01 for TG and CG, respectively. Additionally, S/D ratio of the same artery presented an increase in TG, whereas this ratio diminished in CG (P < 0.01). On Day 8, this parameter showed values of 2.98 ± 0.4 vs 1.62 ± 0.06 for TG and CG, respectively. Resistance index of umbilical artery remained almost unchanged in TG from Day 6 onward, whereas it progressively decreased in CG throughout the course of the study (P < 0.05). On Day 8, RI were 0.89 ± 0.04 and 0.82 ± 0.01, for TG and CG, respectively. Furthermore, on Day 8, S/D ratio of umbilical artery progressively diminished in CG but not in TG (P < 0.01), being 14.7 ± 9.1 vs 5.9 ± 0.3 for TG and CG, respectively. Fetal heart rate was higher in TG than in CG (P < 0.05). Group differences in Doppler parameters appeared on Day 2, when the other clinical or ultrasonographic signs were still absent. It is concluded that blood flow of the uterine and umbilical arteries differed between these normal and abnormal gestations predicting an adverse obstetric outcome. [ABSTRACT FROM AUTHOR]

Contents Matrix metalloproteinase ( MMP) has been implicated as having roles in ovarian folliculogenesis. Here, we determined the expression pattern of six MMPs ( MMP1, MMP2, MMP3, MMP7, MMP9 and MMP13) and their endogenous tissue inhibitor, TIMP1, during cat follicle growth. Different developmental stage follicles were mechanically isolated and gene expression analysed by real-time qPCR while MMP1, 2, 9 and 13 localization was determined by immunohistochemistry. With the exception of MMP13, the amount of MMP mRNA was lowest in primordial follicles and increased thereafter. Peak levels were detected in early antral follicles for MMP1 (72.2-fold increase above primordial follicle amount), MMP2 (10-fold), MMP3 (57-fold) and MMP9 (2.8-fold). MMP7 transcripts increased 2-fold by the primary follicle stage and then plateaued. MMP13 mRNA peaked in primary follicles (2.5-fold) and was lower in more advanced counterparts. TIMP1 sharply increased (6-fold) in secondary follicles and gradually declined in the later stages. MMP1 and MMP9 expression were expressed in the granulosa cells of all follicle stages. MMP2 was immunoreactive in early and antral follicles, especially at granulosa cells adjacent to the antral cavity. By contrast, the MMP13 was weakly detected in primary follicles onward. In summary, there are distinctive and consistent changes in MMPs and TIMP1 expression during follicle development, suggesting that these enzymes play one or more roles in cat folliculogenesis. In particular, high mRNA and protein expression levels of MMP1 and MMP2, especially at the antral stage, indicate that these enzymes likely are involved in antrum formation and expansion. [ABSTRACT FROM AUTHOR]

To test the hypothesis that in domestic cats, postnatal androgens induce sterility, the aims of this study were to describe the reproductive effects and the clinical safety of a postnatal administration of a long term release androgen in this species. Thirteen newborn littermate female kittens were randomly assigned to one of the following treatment groups within the first 24 h of birth: testosterone enanthate 12.5 mg sc (TE; n = 8) or Placebo (PL; n = 5). The animals were subsequently assessed for fecal sexual hormones until puberty was attained and subsequently when matings occurred. After 21 days, ovulation and gestation were diagnosed. All queens were subsequently ovario-hysterectomized. Fecal testosterone concentrations differed between the treatment groups throughout the study period ( P < 0.05) being greater during the first 2 postnatal weeks in those of the TE group ( P < 0.01). Fecal estradiol was not affected by treatment ( P > 0.1). While all the females were receptive during the pubertal estrus ( P > 0.1), two TE (2/8) compared with all (5/5) females of the PL group had ovulations ( P < 0.05). Only one (1/2) compared with three (3/5) of the queens of the TE and PL groups, respectively became pregnant. All kittens of the TE group had transient clitoral enlargement. Anovulatory TE-treated cats had no corpus luteum, and a significant diminution of the endometrial glands as well as of the height of the uterine epithelium. It is concluded that, in domestic cats, a single postnatal supra-physiological dose of testosterone caused a large proportion of queens to be anovulatory and there were also histological endometrial abnormalities that also occurred with this treatment that were accompanied by mild and transient side effects. [ABSTRACT FROM AUTHOR]