bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of Fluorescent Stainings and Flow Cytometry for Canine Semen Assessment
Authors: Niżański W , Partyka A , Rijsselaere T 
Published: December 2012

ContentsOver the last few decades, appreciable progress has been noted in canine semen assessment techniques. The common use of accurate and sensitive diagnostic methods, such as computer‐assisted sperm analysis (CASA), flow cytometry and sperm penetration tests have become routine procedures in specialized andrology laboratories. Many fluorescent probes have been applied to the assessment of specific sperm characteristics in dogs. Flow cytometry enables the observation of cell characteristics such as size, shape and function of the spermatozoon, that can be revealed by a fluorochrome or fluorescent label. The analysis of events detected on dot plots gives accurate and highly reliable information on membrane integrity, acrosomal status, mitochondrial activity, capacitation status, lipid peroxidation, apoptosis and DNA damage. Despite the development of these modern and accurate tools, it is still questionable if the ideal method of semen evaluation, allowing predicting of the fertilizing potential of semen, has been established.
Effect of Refractoriness to Long Photoperiod on Sperm Production and Quality in Tomcats
Authors: Nuñez Favre R , Bonaura MC , Tittarelli CM , Stornelli MC , de la Sota R L , Stornelli MA 
Published: December 2012

ContentsThe aim of this study was to assess whether refractoriness to long photoperiod (LP) could be reversed by subjecting tomcats to a period of short days. Our hypothesis was that photoperiod changes can avoid refractoriness and restore sperm quality and production to that before refractoriness. Tomcats (n = 6) were housed in a conditioned room withLP(12L: 12D) for 45 days of acclimation and then maintained underLPfor 18 month. Then, tomcats were changed to a period of decreasing light at a rate of 8 min/day for 1 month. Tomcats stayed for 1 month with short photoperiod (SP; 8L: 16D) and then were switched back to a period of increasing light at a rate of 8 min/day for 1 month. The experiment was completed after tomcats remained inLPfor 2 months. Toms were anaesthetized and semen samples were collected by electroejaculation every 2 weeks. Sperm parameters were evaluated in all ejaculates, and data were analysed byanova. Motility, velocity, volume, sperm concentration, total sperm count, viability, acrosome integrity, plasma membrane integrity and sperm morphology were higher duringLPcompared with a refractoryLP(p < 0.01). Likewise, velocity, viability, acrosome integrity, plasma membrane integrity and sperm morphology were higher in aLPcompared with aSP(p < 0.05). On the other hand, motility, volume, concentration and total sperm count were similar betweenLPandSP(p > 0.20).Whereas motility, velocity, viability, acrosome integrity and plasma membrane integrity were similar in a refractoryLPcompared withSP(p > 0.05), volume, sperm concentration, total sperm count and sperm morphology were lower in a refractoryLPcompared withSP(p < 0.05). In conclusion, refractoriness and reduced sperm production and quality induced by a prolongedLPof 18 month can be restored after placing tomcats to aSP.
Effect of Natural Photoperiod on Epididymal Sperm Quality and Testosterone Serum Concentration in Domestic Cat (Felis silvestris catus)
Authors: Nuñez‐Favre R , Bonaura MC , Tittarelli CM , Mansilla‐Hermann D , de la Sota RL , Stornelli MA 
Published: December 2012

ContentsThe aim of this study was to assess epididymal sperm characteristics and serum testosterone concentration in cats under natural photoperiod. The hypothesis was that natural photoperiod induces seasonal changes in spermatozoal quality and serum testosterone concentration. Mixed breed tomcats (n = 43) that underwent bilateral orchiectomy at a municipal public pet shelter were used in the study. Epididymides were divided into two groups according to time of castration. In Group I, toms were castrated during increasing light (IL; [winter and spring; n = 24]), and group II, during decreasing light (DL; [summer and fall; n = 19]). Only mature toms castrated in the two lasts weeks of each season were included in this study. Sperm samples were obtained by cutting the cauda epididymis in Tris solution and tested for motility (MOT,% motile), velocity (VEL, 0–5), total sperm cells (TS, 106), acrosome integrity (ACR,% intact; FITC‐PSA), plasma membrane integrity (MI,%intact; CFDA‐PI) and sperm morphology (SM,% normal). Before orchiectomy, blood samples were taken to measure serum concentrations of testosterone (T2) by a solid‐phase RIA. Data were analysed with the mixed procedure of SAS. Toms castrated during IL had higher sperm plasma membrane integrity and better sperm morphology compared to toms castrated during DL (69.0 ± 2.7 vs 60.6 ± 2.1, p < 0.01; 45.9 ± 2.5 vs 35.9 ± 3.4; p < 0.02; respectively) and tended to have higher sperm motility and total number of sperm cells compared to toms castrated during DL (56.3 ± 2.8 vs 47.3 ± 3.7, p < 0.06; 13.8 ± 1.4 vs 10.0 ± 1.8, p < 0.09). However, velocity, acrosome integrity and serum testosterone concentrations were similar between both groups (3.5 ± 0.1 vs 3.4 ± 0.1, p > 0.6; 45.8 ± 3.3 vs 44.0 ± 4.0, p > 0.72; 0.76 ± 0.15 vs 0.59 ± 0.19, p > 0.51; respectively). In conclusion, natural photoperiod induces seasonal changes in sperm quality with a moderate variation in serum testosterone concentrations.
Seasonal Reproductive Activity of Domestic Queens (Felis catus) in the Tropics of Mexico
Authors: Ortega‐Pacheco A , Concha‐Guillermo H , Segura‐Correa J , Jimenez‐Coello M 
Published: December 2012

ContentsThe objective of this study was to determine the seasonal ovarian activity of domestic queens under the tropical conditions of southern Mexico (19° 30′ and 21° 35′ N latitude). A total of 250 reproductive tracts were evaluated, and the frequencies of queens with ovarian activity (oestrus, dioestrus or pregnant), anoestrus, pregnancies and ovulation rate by season of the year (spring, summer, autumn and winter) were calculated. Ovarian activity was observed throughout the year, but a significant reduction occurred during the winter; conversely, anoestrous cases were lower during spring and showed an increase during summer, autumn and winter. Pregnancies were most common during spring and decreased during summer, autumn and winter. Ovulation rate varied from 2.5 to 2.8 during the four seasons. Cystic endometrial hyperplasia, pyometra and ovarian follicular cysts were the most common lesions found. The results demonstrate that domestic queens are not seasonal in tropical conditions, but a clear effect of the season of the year reducing ovarian activity is present.
Increase in Histone Methylation in the Cat Germinal Vesicle Related to Acquisition of Meiotic and Developmental Competence
Authors: Phillips TC , Wildt DE , Comizzoli P 
Published: December 2012

ContentsThis study identified specific changes in histone lysine methylation patterns of the feline germinal vesicle (GV) during pre‐antral‐to‐antral follicle transition, the latter being a key interval for competence acquisition. Oocytes from adult cats were isolated from pre‐antral, early (≤0.5 mm diameter), small (0.6–1 mm) or large (1–3.5 mm) antral follicles and immuno‐stained with anti‐histones H3 trimethylated at lysine 9 (H3K9me3), lysine 4 (H3K4me3), lysine 27 (H3K27me3) or H3 dimethylated at lysine 79 (H3K79me2). The vast majority of oocytes (range, 72.2–85.4%; p > 0.05) contained a GV with H3K9me3 or H3K27me3, regardless of follicular stage/size. However, the proportion of GVs with H3K4me3 or H3K79me2 was higher in early antral follicles (42.6%; p < 0.05) compared with other stages (range, 12.1–15.2%). Therefore, H3K4me3 and H3K79me2 (both known to be associated with selective gene activation) appear to be reliable markers of onset of GV competence during the pre‐antral‐to‐antral transition phase. By contrast, H3K9me3 and H3K27me3 (both known to be related to selective gene repression) seem more linked to expression patterns during the GV stage and are less useful indicators during the entire folliculogenesis interval.
Applying Embryo Cryopreservation Technologies to the Production of Domestic and Black‐Footed Cats
Authors: Pope CE , Gómez MC , Galiguis J , Dresser BL 
Published: December 2012

ContentsOur objectives were (i) compare in vitro development of early cleavage stage domestic cat embryos after cryopreservation by minimal volume vitrification vs a standard slow, controlled‐rate method, (ii) determine viability of vitrified domestic cat embryos by oviductal transfer into synchronous recipients and (iii) evaluate in vivo survival of black‐footed cat (BFC, Felis nigripes) embryos after intra‐ and inter‐species transfer. In vitro‐derived (IVM/IVF) cat embryos were used to evaluate in vitro development after controlled‐rate cryopreservation vs vitrification vs controls. Blastocyst development was similar in both groups of cryopreserved embryos (22–26%), but it was lower (p < 0.05) than that of fresh embryos (50%). After embryo transfer, four of eight recipients of vitrified embryos established pregnancies – three of six (50%) and one of two (50%) that received embryos from in vivo‐ and in vitro‐matured oocytes, respectively. Three male and two female kittens weighing from 51 to 124 g (mean = 88 g) were delivered on days 61–65 of gestation. In BFC, four intra‐species embryo transfer procedures were carried out – two recipients received fresh day 2 embryos (n = 5, 8) and two recipients received embryos that had been cryopreserved on day 1 (n = 6) or 2 (n = 8). A 2–year‐old recipient of cryopreserved embryos established pregnancy and delivered two live male kittens. Subsequently, five cryopreserved BFC embryos were transferred to a domestic cat recipient. On day 29, the recipient was determined to be pregnant and delivered naturally a live, healthy female BFC kitten on day 66. In summary, in vivo survival of vitrified domestic cat embryos was shown by the births of kittens after transfer into recipients. Also, we demonstrated that sperm and embryo cryopreservation could be combined with intra‐ and inter‐species embryo transfer and integrated into the array of assisted reproductive techniques used successfully for propagation of a rare and vulnerable felid species, the black‐footed cat.
Chromatin Patterns of Immature Canine Oocytes after In Vitro Maturation
Authors: Reynaud K , Chebrout M , Tanguy‐Dezaux C , de la Villéon G , Chastant‐Maillard S 
Published: December 2012

ContentsIn canine species, in vitro maturation (IVM) rates of oocytes collected from anoestrous ovaries are low (<20%). Several IVM media have been tested without significant improvements. A critical step in the evaluation of culture conditions is the observation of the meiotic stage reached by the oocytes. The present study was designed to investigate the chromatin patterns of in vitro matured oocytes by visualizing Germinal Vesicle (GV) and Germinal Vesicle Breakdown (GVBD) structures at 72 h of IVM. Nuclear stages of 1678 oocytes were evaluated by confocal microscopy after IVM. 1204 oocytes were non‐degenerated, and 94.4% were still immature and at GV stage. Five different patterns of chromatin configuration were observed. Higher percentages of oocytes with unmodified GV and with diffuse (58%; Type A) and filamentous chromatin (19%; Type B) were observed in comparison with those with modifications in the GV such as patched chromatin (12.5%; Type C), surrounded‐nucleolus (3%; Type D) and in vivo type chromatin/fully grouped chromatin (2.5%; Type E). These results indicate that GVBD (absence of nucleolus, nucleus breakdown) is rarely observed in vitro. The percentage of type C‐D‐E GVs and MI (meiotic resumption) and of MII (completion of meiosis) can be used to evaluate meiotic resumption after IVM. Our results indicate that although a low number of in vitro matured oocytes exhibit the chromatin configurations observed in in vivo collected oocytes, chromatin changes in the GV can be induced during IVM.
Folliculogenesis, Ovulation and Endocrine Control of Oocytes and Embryos in the Dog
Authors: Reynaud K , Fontbonne A , Saint‐Dizier M , Thoumire S , Marnier C , Tahir MZ , Meylheuc T , Chastant‐Maillard S 
Published: December 2012

ContentsReproductive physiology in dogs is quite unusual compared with that in other mammalian species. The peculiarities include the presence of numerous polyoocyte follicles, the ovulation of an immature oocyte (GV stage, non‐fertilizable) and a peri‐ovulatory period during which concentrations of circulating progesterone are particularly high. The aim of this review is to examine the unusual aspects of the reproductive physiology of dogs and how this relates to the clinical biology of this species.
Histological Characterization and Immunohistochemical Localization of Ki‐67 and VEGF Factors in Bitches' Corpus Luteum at Cyclic and Early and Late Gestational Diestrous
Authors: Ribeiro AA , Martin I , Derussi AAP , Ferioli RB , Laufer‐Amorim R , Lopes MD 
Published: December 2012

ContentsThe luteal phase on pregnant and non‐pregnant bitches is characteristic of this species and resembles significantly with respect to the growth pattern and luteal regression. Histological and immunostaining studies of the corpus luteum (CL) may help to elucidate differences between the CL of pregnant and non‐pregnant bitches. The purpose of this study was to characterize histologically and localize by immunohistochemistry the cell proliferation (Ki‐67) and vascular endothelial growth (VEGF) factors in the CL of pregnant and non‐pregnant bitches. Eighteen bitches were analysed and distributed into three groups: In group I (gestational diestrous), seven bitches were subjected to two inseminations at 4 and 6 days after the pre‐ovulatory LH surge and ovariohysterectomized (OSH) at 8–21 days after the first insemination. In group II (cyclic diestrous; control), 6 (Ki‐67) or 8 (VEGF) bitches that were determined as non‐pregnant were OSH at 12–25 days of the pre‐ovulatory LH surge. In group III (late pregnancy), three bitches had their ovary removed during caesarean section at 62–64 days after the pre‐ovulatory LH surge. Portions of the ovarian cortex containing CLs were cut and stored for histological and immunohistochemical analysis. Histological evaluation of the ovarian cortex showed a marked similarity in the morphological pattern among the CLs in all three groups. The morphology and expression pattern of VEGF and Ki‐67 factors in CLs of cyclic and gestational diestrous bitches were similar but significantly lower than that of late pregnant bitches (p < 0.05).
Computer‐Assisted Sperm Analysis in Dogs and Cats: An Update after 20 Years
Authors: Rijsselaere T , Van Soom A , Maes D , Nizanski W 
Published: December 2012

ContentsIn dogs and cats, computer‐assisted sperm analysis (CASA) was originally described almost 20 years ago. Subsequently, numerous CASA systems were validated and used for various applications in dogs and to a lesser extent in cats. CASA systems offer an accurate, rapid, objective and simultaneous assessment of different semen parameters allowing the visualization of subtle changes in sperm characteristics, which cannot be identified by conventional semen analysis. The main problems of these computerized measuring devices are the relatively high investment costs and the need for standardization and validation before any practical use is possible. In comparison with automated motility and concentration assessment, automated morphometry and morphology assessment is more complex and time‐consuming. Once validated, CASA systems can be routinely used in veterinary centres for assessment of fertility and for the improvement of sperm diluters, cooling and cryopreservation procedures in dogs and cats. Furthermore, information obtained by CASA systems could also be important when monitoring for example the effect of environmental stress on spermatozoa and for toxicity studies. In cats, CASA is less documented, and most studies describe the characteristics of epididymal sperm, which is frequently used for in vitro fertilization in cats. Implementation of the CASA technique in cat reproduction could be interesting to further optimize assisted reproductive techniques in domestic cats and endangered wild felids.
Semen Quality and Onset of Sterility Following Administration of a 4.7‐mg Deslorelin Implant in Adult Male Dogs
Authors: Romagnoli S , Siminica A , Sontas BH , Milani C , Mollo A , Stelletta C 
Published: December 2012

ContentsThe purpose of this study was to define (i) the interval between treatment and sterility, and (ii) semen quality in male dogs administered a 4.7‐mg deslorelin implant. Six healthy, adult dogs of various breeds and body weights were implanted with deslorelin (Suprelorin, Virbac) and followed every 2 weeks with semen and blood collections. Semen quality remained stable or even improved during the first month following treatment and then showed a progressive decline until the end of the study, except for sperm morphology, which was unaffected by the treatment. Complete sterility was achieved on post‐treatment days 70, 84, 60, 23, 51 and 40 for dogs 1 to 6, respectively. The 4.7 mg deslorelin implant caused a significant (p < 0.05) decrease in serum testosterone as well as sperm motility. Our results (i) confirm the efficacy of deslorelin in causing reversible sterility in male dogs, (ii) confirm and provide details about endocrine and seminal parameters involved in this process and (iii) contribute to define the interval between treatment and achievement of complete sterility. Practitioners should be aware that such interval may be longer than 2 months in some cases, and that fertility may actually be increased during the first 2–4 weeks post‐treatment.
B‐mode and Contrast‐Enhanced Ultrasonographic Findings in Canine Prostatic Disorders
Authors: Russo M , Vignoli M , England GCW 
Published: December 2012

ContentsUltrasound is a common imaging technique used for examination of the dog's prostate gland that has proven to be successful for diagnosis of many clinical conditions. Whilst B‐mode ultrasound is useful for assessment of the prostate gland, there appears to be substantial variation in the appearance of some pathological conditions, although individual experienced ultrasonographers often have a high aptitude for making a correct diagnosis. Here, we report the normal volume and appearance of the canine prostate gland with B‐mode ultrasound and use thematic analysis to identify the categories described by experienced ultrasonographers when reporting both normal and abnormal findings. Four thematic categories were identified: background echotexture, parenchymal stippling, generalized appearance and focal changes. We found that a relatively narrow variety of thematic variables (descriptors) were used for reporting of prostatic pathology within these thematic categories. There was also poor association between these descriptors and the underlying pathology, with the only unique descriptors being ‘cysts’ for benign prostatic hyperplasia and ‘parenchymal mineralization’ for adenocarcinoma. In comparison with the limitations of B‐mode ultrasound, we document the value of measurement of vascular perfusion kinetics using contrast‐enhanced ultrasound and demonstrate how perfusion kinetics differ between benign and malignant prostatic disease.
Cellular Characteristics of Cultured Canine Trophoblasts
Authors: Sahlfeld L , Hazzard T , Kutzler M 
Published: December 2012

ContentsShallow trophoblast invasion is detrimental in human pregnancies, but represents normal endotheliochorial placentation in dogs. Factors regulating shallow trophoblast invasion into the canine decidua are not well described, but it is known that matrix metalloproteinases (MMPs) play a crucial role in trophoblast invasion in many species. Following the methods previously described for isolating human trophoblasts, canine trophoblasts were isolated using collagenase and trypsin digestions with Percoll density gradient centrifugation. In addition, placental pieces were cryopreserved prior to primary culture following methods previously described for human tissue. Expression of cytokeratin‐7, MMP2 and MMP9 was confirmed using fluorescent immunocytochemistry. Cellular morphology was similar to that reported for trophoblasts. More than 97% of the cells cultured expressed cytokeratin‐7. More cultured canine trophoblasts expressed MMP9 (54.7 ± 3.4%) compared with MMP2 (40.3 ± 1.8%) (p = 0.02). Although both MMPs were immunolocalized to the cytoplasm, MMP2 was found in large, coalescing granules, whereas MMP9 was more diffusely expressed throughout the cell. Cryogenic freezing of placental tissue prior to primary cell culture had no effect on cell proliferation (p = 0.37). This research has established a baseline for future studies investigating the canine placenta as a model for disorders of shallow trophoblast invasion in humans.
Generation and Characterization of Phage‐GnRH Chemical Conjugates for Potential Use in Cat and Dog Immunocontraception
Authors: Samoylov A , Cox N , Cochran A , Wolfe K , Donovan C , Kutzler M , Petrenko V , Baker H , Samoylova T 
Published: December 2012

ContentsOverpopulation of cats and dogs is a serious worldwide problem that demands novel, safe and cost‐effective solutions. The objective of this study was to generate and characterize phage‐peptide conjugates with gonadotropin‐releasing hormone (GnRH) for potential use as an immunocontraceptive. A filamentous phage vector f5‐8 with wild‐type phage coat proteins was used as a carrier for construction of chemical conjugates with GnRH, a peptide that acts as a master reproductive hormone. In such conjugates, the phage body plays the role of a carrier protein, while multiple copies of GnRH peptide stimulate production of neutralizing anti‐GnRH antibodies potentially leading to contraceptive effects. To generate the constructs, four different GnRH‐based peptides were synthesized and conjugated to phage particles in a two‐step procedure: (i) peptides were reacted with phage to form a conjugate using 1‐ethyl‐3‐[3‐dimethylaminopropyl]carbodiimide hydrochloride chemistry (EDC) and (ii) the conjugates were separated from remaining free peptides by dialysis. Formation and specificity of phage‐GnRH conjugates were confirmed by three independent methods: spectrophotometry, electron microscopy and ELISA. When the conjugates were tested for interaction with sera collected from cats and dogs immunized with GnRH‐based vaccines in independent studies, strong specific ELISA signals were obtained, suggesting the potential use of the conjugates for cat and dog immunosterilization. The ability of the conjugates to stimulate production of anti‐GnRH antibodies in vivo was evaluated in mice. While optimization of dose, immunization route and adjuvant still requires investigation, our preliminary results demonstrated the presence of anti‐GnRH antibodies in sera of mice immunized with such conjugates. Fertility trials in cats and dogs will be needed to evaluate contraceptive potentials of the phage‐GnRH peptide chemical conjugates.