bluuebunny/crossref_metadata_2025_split huggingface dataset:
Capabilities and Challenges of Examination of Gene Expression for Quality Assessment of Domestic Cat Embryos
Authors: Hribal R , Braun BC , Ringleb J , Jewgenow K 
Published: December 2012

ContentsEarly embryos are characterized by an accurately controlled gene expression pattern that might be deregulated during in vitro culture (IVC). The expression pattern of the developmental genes may serve as markers for embryo quality. Here, we examined the temporal pattern of relative mRNA abundance of genes important for early embryonic development in embryos produced by different fertilization methods [in vitro fertilization (IVF) vs intracytoplasmic sperm cell injection (ICSI)] and sperm sources (fresh vs frozen–thawed) applying reverse transcriptase (RT) PCR. The temporal pattern of gene expression was found to be gene specific and similar in all four examined groups in a semi‐quantitative assay. In morulae, higher relative mRNA levels were found in embryos generated with fresh sperm, whereas in blastocysts, mRNA abundance tended to be higher in embryos produced with cryopreserved sperm cells. This indicates an influence of sperm cryopreservation on the temporal gene expression pattern in early cat embryos. We also examined relative mRNA abundances by real‐time quantitative RT‐PCR in blastocysts. In this context, blastocysts produced with fresh semen tended to have lower DNA methyltransferase 3A (DNMT3A) but higher gap junction protein alpha 1 (GJA1) and octamer‐binding transcription factor 4 (OCT4) mRNA levels compared with those derived with frozen–thawed semen. We conclude that assessing embryo quality by measuring gene expression pattern in early embryos is challenging because of a high variability between individual embryos.
Life Cycle of Feline Corpora lutea: Histological and Intraluteal Hormone Analysis
Authors: Jewgenow K , Amelkina O , Painer J , Göritz F , Dehnhard M 
Published: December 2012

ContentsThe corpus luteum (CL) is a transient hormone gland on the ovary that produces progesterone (P4) for the maintenance of pregnancy. It develops from residual follicular granulosa and theca cells after ovulation. Very little is known about the cellular and hormonal processes within CLs obtained from pregnant and pseudopregnant felids. Therefore, our aim was to review the luteal function in feline CLs of different reproductive stages in conjunction with our data obtained in domestic cats and Eurasian lynxes. Corpus luteum function in lynxes is of particular interest, as a post‐partum luteal activity was suggested based on repeated ultrasonography and endocrine examinations. Histology of CL from pregnant and pseudopregnant domestic cats clearly reflects the luteal function. The formation of the CL after ovulation is characterized by transforming of theca and granulosa cells into steroidogenic luteal cells and is accompanied by increased intraluteal and circulating P4 levels. Luteal regression is steadily progressive; the first signs (coarsed vacuolization, increased proportion of non‐steroidogenic cells) are visible already in CL from the second trimester of pregnancy.
Cytokines as Immunological Markers for Systemic Inflammation in Dogs with Pyometra
Authors: Karlsson I , Hagman R , Johannisson A , Wang L , Karlstam E , Wernersson S 
Published: December 2012

ContentsPyometra is a disease in dogs caused by bacterial infection of the uterus and resulting in SIRS (systemic inflammatory response syndrome) in nearly 6 of 10 cases. Clinical diagnostic criteria for SIRS are relatively unspecific, and biomarkers for the diagnosis of pyometra and SIRS in dogs are needed. Serum samples from 32 dogs were used in this study and grouped into dogs with pyometra and SIRS, dogs with pyometra without SIRS and healthy controls. The serum concentrations of IFN‐γ, IL‐4, IL‐6, IL‐7, IL‐8, IL‐10, IL‐15, IL‐18 and TNF‐α were measured using multiplex analyses. The serum concentrations of CRP (C‐reactive protein) were determined using sandwich ELISA. IL‐7, IL‐8, IL‐15, IL‐18 and TNF‐α were detected in >94% of samples. IL‐10 was detected in 28% of samples, and IL‐4, IL‐6 and IFN‐γ were undetectable. Higher serum concentrations of IL‐7 (p < 0.05) were detected in SIRS‐positive dogs with pyometra (n = 13) as compared with healthy controls (n = 11). The concentrations of IL‐8 were higher in SIRS‐positive dogs with pyometra compared to the SIRS‐negative group (n = 8; p < 0.05). Positive correlations of IL‐15 with IL‐18 (p < 0.0001) and with the concentrations of IL‐7 (p < 0.0001 for both) were found, although there was no significant difference between groups. Furthermore, IL‐15 correlated with concentrations of CRP (p < 0.05), which were higher in dogs with pyometra compared to controls (p < 0.0001). Our data suggest a role of several cytokines in the development of a systemic disease in dogs with pyometra and a possible diagnostic value for serum CRP, IL‐7, IL‐15 and IL‐18 in canine SIRS caused by pyometra.
Species‐specific Challenges in Dog Cloning
Authors: Kim GA , Oh HJ , Park JE , Kim MJ , Park EJ , Jo YK , Jang G , Kim MK , Kim HJ , Lee BC 
Published: December 2012

ContentsSomatic cell nuclear transfer (SCNT) is now an established procedure used in cloning of several species. SCNT in dogs involves multiple steps including the removal of the nuclear material, injection of a donor cell, fusion, activation of the reconstructed oocytes and finally transfer to a synchronized female recipient. There are therefore many factors that contribute to cloning efficiency. By performing a retrospective analysis of 2005–2012 published papers regarding dog cloning, we define the optimum procedure and summarize the specific feature for dog cloning.
Endocrine and Molecular Control of Luteal and Placental Function in Dogs: A Review
Authors: Kowalewski MP 
Published: December 2012

ContentsIn the domestic dog (Canis familiaris), the corpus luteum (CL) is the only source of progesterone (P4) in non‐pregnant and pregnant animals. The progesterone secretion profiles are almost identical in both conditions until the last third of the luteal phase when the gradual P4 decline turns into a steep drop in pregnant bitches, indicating the onset of parturition. Consequently, the length of the CL‐phase in non‐pregnant dogs exceeds the luteal lifespan in pregnant animals. The canine CL‐function is regulated by many species‐specific regulatory mechanisms, the most intriguing of which is the reported independence of gonadotropic support during the first third of dioestrus. Recently, PGE2 has been proposed as one of the most important luteotropic factors acting locally during this time, but afterwards prolactin (PRL) appears to be the main luteotropic factor. Luteal regression/luteolysis occurs, however, in spite of an increased gonadotropic support. Lately, by demonstrating the expression of PRL‐receptor (PRLr), a new insight into possible regulatory mechanisms has indicated that the supply of P4 could be controlled upstream of the steroidogenic machinery at the level of PRLr expression and/or function, subsequently leading to the functional suppression of the steroidogenic machinery. An endogenous source of a luteolytic agent is apparently lacking, implicating the luteal regression in non‐pregnant bitches as a passive, degenerative process even if the PGF2α‐receptor is constitutively expressed in canine CL. This is in contrast to pregnant dogs in which prepartum luteolysis seems to be an active process of CL destruction by PGF2α of utero/placental origin targeting the luteal PGF2α‐receptor.
Who Let the Dogs In: A Canine Trophoblast Invasion Model for Pre‐eclampsia
Authors: Kutzler M , Sahlfeld L , Fellows E 
Published: December 2012

ContentsPre‐eclampsia affects 2–8% of pregnant women worldwide and is the third leading cause of maternal mortality in the United States, accounting for 20% of maternal deaths, for which the only known cure is delivery of the placenta. It is known that pre‐eclampsia results from defects within the trophoblast invasion of the endometrium and myometrium. At a morphological level within the pre‐eclamptic human placenta, trophoblast invasion is shallow, and this results in hypoperfusion, which is a life‐threatening condition for both the mother and the foetus. Pre‐eclampsia has been intensively investigated for over 50 years, and yet the causes are largely unknown. Despite a large body of data, it is still unknown exactly which mechanisms regulate trophoblast invasion. An effective animal model may be crucial to understanding the underlying causes of pre‐eclampsia. A canine model is a proposed improvement on the current efforts to investigate disorders of shallow trophoblast invasion throughout gestation and to improve understanding of the factors that regulate trophoblast invasion. The objectives of this research were to elucidate and compare cellular and molecular similarities between normal canine trophoblasts with those from recently published reports on pre‐eclampsia in women.
Comparative Fertility of Freshly Collected vs Frozen–Thawed Semen with Laparoscopic Oviductal Artificial Insemination in Domestic Cats
Authors: Lambo CA , Grahn RA , Lyons LA , Bateman HL , Newsom J , Swanson WF 
Published: December 2012

ContentsArtificial insemination (AI) is potentially invaluable as an adjunct to natural breeding for the conservation management of non‐domestic felid populations. The efficacy of AI, however, must be substantially improved for applied use, especially when using frozen semen. Our recent advances in using laparoscopic oviductal AI (LO‐AI) with low sperm numbers and freezing of cat semen in a soy lecithin–based cryoprotectant medium suggest that combining these two approaches might improve pregnancy outcomes with frozen–thawed spermatozoa. In this study, our objectives were to (i) assess the effect of two gonadotropin dosages (100 vs 150 IU eCG) on ovarian response in domestic cats and (ii) compare the relative fertility of frozen–thawed and fresh semen in vivo following LO‐AI. All 16 females ovulated after gonadotropin treatment and were inseminated with fresh semen from one male and frozen–thawed semen from a second male. There were no differences between gonadotropin dosages in CL number, pregnancy percentage or litter size. Half (8/16) of the females conceived, with seven females giving birth to a total of 36 offspring. Paternity analysis showed that more kittens resulted from LO‐AI with fresh (28/36, 78%) than frozen–thawed (8/36, 22%) semen, possibly due to impaired motility and longevity of thawed sperm. These results demonstrated that viable offspring can be produced by AI using semen frozen in a soy lecithin–based medium. Insemination with greater numbers of frozen–thawed spermatozoa, combined with further refinement of cat sperm cryopreservation methods, may be necessary to optimize pregnancy success with LO‐AI in domestic and nondomestic cats.
Evaluation of Sperm DNA Peroxidation in Fertile and Subfertile Dogs
Authors: Lopes‐Santiago BV , Monteiro GA , Bittencourt R , Arduino F , Ovidio PP , Jordão‐Junior AA , Araújo JP , Lopes MD 
Published: December 2012

ContentsOxidative stress (OS) has been recognized as one of the most important causes of male infertility. The antioxidant activities of seminal plasma and epididymal fluid are not enough to prevent OS, which can damage sperm membranes and DNA, so antioxidant supplementation has been used as a treatment of male infertility. The aim of this experiment was to evaluate the DNA peroxidation before and after antioxidant supplementation with vitamin C and E in dogs with and without fertility problems. A total of eleven dogs were used and were divided in two groups: fertile group (G1), dogs with normal spermiogram (n = 5); subfertile group (G2): dogs with low sperm count (<20 × 106 sptz/ml) and/or more than 30% of total sperm pathology (n = 6). Both groups received 500 mg/day of vitamin C and 500 mg/day of vitamin E for 60 days. A semen sample was collected before (M1) and after (M2) oral supplementation. Samples were analysed for DNA peroxidation by measuring the 8‐hydroxy‐2′‐deoxyguanosine concentration. No significant difference was observed between groups at either time. Oral supplementation with 500 mg/day of vitamin C and 500 mg/day of vitamin E did not change the DNA peroxidation in fertile and subfertile dogs.
Cryosurvival of Ex Situ and In Situ Feline Oocytes
Authors: Luvoni GC 
Published: December 2012

ContentsCryosurvival of feline oocytes preserved as isolated cells (ex situ) or enclosed in ovarian follicles (in situ) has been demonstrated, and significant advances have recently been achieved. However, an ideal protocol for oocyte cryopreservation has not been established to date because of extreme sensitivity of the structural complex to chilling injury. Several factors, such as stage of maturation, membrane permeability and plasticity of the cytoskeleton, affect cryosurvival of the oocyte. Also, intercellular communications between cumulus cells and oocyte are compromised after freezing or vitrification of ex situ or in situ cumulus–oocyte complexes, which has a detrimental effect on oocyte maturational competence. Despite these issues, embryo development, pregnancies and live kittens have been obtained after in vitro fertilization (by ICSI) and transfer of embryos derived from cryopreserved oocytes. It is a general belief that the efficiency of cryopreservation would increase through a better understanding of oocyte responses to cryoprotectants, cooling rates and all the physical events occurring during the exposure of feline oocytes to low temperatures. Cryobanking of feline oocytes would significantly contribute to the preservation of rare genotypes and to the maintenance of a valuable source of genetic material for research applications.
Results of GnRH Agonist Implants in Oestrous Induction and Oestrous Suppression in Bitches and Queens
Authors: Maenhoudt C , Santos NR , Fontaine E , Mir F , Reynaud K , Navarro C , Fontbonne A 
Published: December 2012

ContentsOver the last 10–15 years, long‐acting GnRH agonists have become widely available. In the field of small animal reproduction, most recent studies have focused on the use of two compounds developed under the form of subcutaneous implants: azagly‐nafarelin and deslorelin. Only the latter has been commercially available for use in male dogs, first in Australia and New Zealand, then in several countries of the European Union since 2008. Although officially marketed for male dogs, this compound has also been studied in bitches and more recently in queens. Some published papers or recent presentations at congresses – still unpublished – have focused on the use of GnRH agonists implants in females.
Comparison of Two Different Extenders for Cryopreservation of Epididymal Dog Sperm
Authors: Martins MIM , Justino RC , Sant'Anna MC , Trautwein LGC , Souza FF 
Published: December 2012

ContentsThe collection of epididymal sperm is an option for preservation of germplasm of genetically superior animals that need to be orchiectomized or have died. The extender type used to freeze sperm is important to avoid spermatozoal membrane damage and to preserve semen quality after cryopreservation. The objective of this study was to verify the effects of a commercial bovine extender (Bovimix®; Nutricell, Campinas) and a traditional TRIS–citric acid–glucose–egg yolk–7% glycerol extender on cryopreservation of canine epididymal sperm. The testes of 13 adult dogs were kept at 5°C for 24 h in saline solution, and epididymal sperm was recovered in Ringers solution without lactate and were evaluated for motility. Samples with ≥80% motility were pooled and then divided before dilution and packaging in 0.5 ml plastic straws, equilibration at 4°C for 1 h, freezing in nitrogen vapour for 20 min and storing at −196°C. The straws were thawed at 56°C for 10 s and were evaluated for motility by computer assisted analysis (CASA). The semen parameters, sperm movement index, linearity, total motility and rapid progressive motility were statistically higher in Bovimix® than TRIS. In contrast, amplitude of lateral head displacement, slow sperm and static sperm were lower in Bovimix®. Despite the high percentage of sperm defects in epididymal cells, regardless of the extender, we concluded that Bovimix® is a viable alternative for the freezing of canine epididymal sperm.
Application of Genomic and Molecular Methods to Fundamental Questions in Canine and Feline Reproductive Health
Authors: Meyers‐Wallen VN 
Published: December 2012

ContentsMolecular tools are becoming increasingly available to investigate the genetic basis of reproductive disorders in dogs and cats. These were first successful in identifying the molecular basis of diseases inherited as simple Mendelian traits, and these are now being applied to those that are inherited as complex traits. In order to promote similar studies of reproductive disorders, we need to understand how we can play a proactive role in accumulating sufficient case material. We also need to understand these mutation discovery tools and identify collaborators who have experience with their use. The candidate gene and genomic approaches to mutation discovery in dogs are presented, including new sequencing methods and those used to confirm that a mutation has a role in disease pathology. As the final goal is to use our study results to prevent inherited disorders, we need to consider how we can promote efficiency in obtaining DNA test results and providing genetic counselling.
Vitrification of Domestic Cat Oocytes – Effect on Viability and Integrity of Subcellular Structures
Authors: Mikołajewska N , Müller K , Niżański W , Jewgenow K 
Published: December 2012

ContentsCryopreservation of female gametes is a crucial part of assisted reproduction techniques. The domestic cat is a model for both wild felids and human research. The aim of this study was to evaluate the effect of different vitrification protocols applied to feline oocytes at different maturational stages on the preservation of oocyte viability and integrity of subcellular structures. The vitrification solutions consisted of 20% ethylene glycol, 20% DMSO, 20% FCS, and 1.5 m Trehalose with and without 10% Ficoll PM‐70. Markers for cell viability (PI/FDA), cytoskeleton organization (Anti‐α‐Tubulin–FITC antibody, Phalloidin‐TRITC), as well as nuclear configuration (DAPI) were used for evaluation of vitrified‐warmed oocytes. Our results show that 52% and 41% of live mature and immature oocytes, respectively and until 32% of microtubules, 28% of nuclear configuration and 36% of microfilaments in the normal pattern can be obtained with protocol described in this paper. According to our data, Ficoll PM‐70 essentially improves the oocytes survival upon vitrification.
Progesterone Receptor Isoforms in the Mammary Gland of Cats and Dogs
Authors: Gracanin A , de Gier J , Zegers K , Bominaar M , Rutteman GR , Schaefers‐Okkens AC , Kooistra HS , Mol JA 
Published: December 2012

ContentsProgesterone exerts its effect by binding to specific progesterone receptors (PR) within the cell. In dogs and cats, no data are available on PR isoforms as found in other species. We therefore investigated the sequence of the PR gene and encoded protein in dogs and cats, the expression of PR isoforms in mammary tissue using Western blots and the presence of PR in mammary tissue using immunohistochemistry. Comparison of the amino acid sequence of the canine and feline PR with human PR revealed major differences in the PR‐B‐specific upstream segment (BUS). However, the essential activation function 3 (AF3) domain was intact in the cat but mutated in the dog. The DNA and ligand‐binding domains were highly similar among the species. In cats with fibroadenomatous hyperplasia (FAH), high expression of PR mRNA together with growth hormone (GH), GH receptor (GHR) and IGF‐I mRNA was found in comparison with feline mammary carcinomas. Immunohistochemical analysis showed strong nuclear as well as cytoplasmic staining for PR in FAH. Western blot analysis revealed expression of the PR‐A and PR‐B isoforms in the feline mammary gland. In canine mammary tissue, the most abundant PR staining was found in proliferative zones of the mammary gland. Western blot analyses showed mainly staining for PR‐A with lower PR‐B staining. It is concluded that in dogs and cats both PR isoforms are expressed. The role of mutations found in the canine PR‐B is discussed.
Embryonic Diapause: Advances in Understanding the Enigma of Seasonal Delayed Implantation
Authors: Murphy BD 
Published: December 2012

ContentsEmbryonic diapause is an evolutionary strategy by which a reversible arrest in embryo development occurs. In its two forms, facultative and obligate, it assures that offspring are born when optimal maternal and environmental conditions are present to ensure maximal survival. We have explored obligate delayed implantation in the mink (Neovison vison) over four decades: first by evaluation of the environmental regulation, then by determination of the pituitary factors that maintain diapause and provoke implantation followed by exploration of the ovarian contribution to the process. As the uterine environment is the proximal regulator of diapause, we employed a strategy of global gene analysis to discover differentially expressed pathways during embryo arrest and reactivation. These trials revealed that the synthesis of polyamines was increased in the uterus with reactivation of the embryo in vivo. Subsequent experiments demonstrated that the polyamine, putrescine, was capable of inducing escape of the embryo from obligate diapause, providing strong evidence that the paucity of polyamines induces developmental arrest, and reactivation is coupled to renewed uterine and/or embryonic synthesis of these polycations.
Source of Protein Supplementation during In Vitro Culture does not Affect the Quality of Resulting Blastocysts in the Domestic Cat
Authors: Nestle E , Graves‐Herring J , Keefer C , Comizzoli P 
Published: December 2012

ContentsThe objective of this study was to assess and compare the quality of cat blastocysts produced in vitro using commercial blastocyst growth media supplemented with different sources of proteins (serum protein substitute from in vitro maturation through embryo development vs 4 mg/ml of bovine serum albumin for maturation and 5% foetal calf serum for fertilization and embryo development). Impact was specifically examined on the proportion of blastocyst formation, total number of blastomeres, proportion of inner cell mass and expression of pluripotency marker proteins NANOG and OCT‐4. Blastocyst formation per total cleaved embryos was similar (p > 0.05) regardless of the protein supplementation. There were no differences (p > 0.05) between culture conditions regarding average number of blastomeres and proportion of inner cell mass in each embryo. Presence of OCT‐4 protein was detected in nuclei of both trophectoderm and inner cell mass region, with a stronger signal in the latter regardless of the culture medium. NANOG protein also was present in the inner cell mass regardless of the in vitro culture condition. We therefore demonstrated that serum protein substitute was as good as semi‐defined protein sources for the production of good‐quality blastocysts and embryonic stem cells. In addition, a single defined medium could be successfully used for cat oocyte maturation, in vitro fertilization and embryo development.