bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Mitochondrial and Actin Patterns in Cat Oocytes and Blastocysts
Authors: González R , Gómez MC , Pope CE , Brandt YCB
Published: December 2012

Contents

Actin microfilaments and mitochondria distribution are considered useful markers of cytoplasmic maturation, but no information is available regarding their distribution in cat oocytes and embryos. Thus, the purpose of this study was to (i) assess cytoplasmic characteristics of the oocyte by mitochondria and actin staining in immature and in vitro/in vivo matured cat oocytes and (ii) characterize mitochondria and actin distribution in in vitro produced blastocysts by confocal laser scanning microscopy. Additionally, in vivo matured oocytes were collected to assess mitochondria and actin. Transzonal cumulus cell projections were more abundant in immature oocytes than in matured oocytes. A relocation of mitochondria throughout meiosis was not clearly observed. However, most in vitro produced blastocysts were of good quality, according to their actin cytoskeleton integrity and mitochondria distribution. The functional significance of mitochondria distribution in cat oocytes in relation to their developmental competence requires further research. This study represents the original description of actin and mitochondrial patterns in cat oocytes and embryos.
Serum Progesterone in Pregnant Bitches Supplemented with Progestin Because of Expected or Suspected Luteal Insufficiency
Authors: Günzel‐Apel A , Urhausen C , Wolf K , Einspanier A , Oei C , Piechotta M
Published: December 2012

Contents

Progesterone profiles of individual bitches may vary considerably both between and within individuals during pregnancy and non‐pregnancy. Suspected luteal deficiency is commonly purported but is difficult to evaluate in clinical cases when progesterone is supplemented because this masks the underlying hormone changes. Therefore, in this study, suspected cases of luteal deficiency (six pregnancies from five bitches) were supplemented with oral medroxyprogesterone acetate (MPA), allowing measurement of endogenous progesterone using conventional assay. MPA (0.1 mg/kg) treatment commenced between days 30 and 36 after estimated ovulation and was continued for 18–28 days. Endogenous progesterone was measured throughout treatment, and blood was additionally analysed for prolactin (PRL) and relaxin (RLN) as well as MPA. The latter revealed delayed MPA clearance in two bitches, in which Caesarean operation had to be performed because of a low foetal heart rate. In two cases with confirmed basal concentrations of both P4 and MPA at term, spontaneous parturition occurred. Low endogenous progesterone during pregnancy was not apparent in three bitches that had previously had a short inter‐oestrous interval of which two had previously had confirmed short luteal phase. However, in the remaining two cases, there had been previous pregnancy failure, but in only one of these, a premature decrease in endogenous progesterone to <2 ng/ml was detected. The latter had also low concentrations of PRL and RLN. The results of this preliminary clinical study suggest that abnormal progesterone profiles in pregnancy may be uncommon in bitches even when there has been previously documented short inter‐oestrous interval. However, luteal deficiency may be suspected in bitches with a history of repeated pregnancy failure or abortion. MPA supplementation appears to be efficacious for management of suspected luteal deficiency and verification of the ovarian dysfunction, but care should be taken regarding the timing of MPA withdrawal and prolongation of pregnancy because of delayed elimination of MPA from blood circulation.
Recombinant Feline Oviductin – A Powerful Tool for Functional IVF Studies in the Domestic Cat
Authors: Hachen A , Jewgenow K , Krause E , Braun BC
Published: December 2012

Contents

Oviductins are highly conserved proteins that seem to play an important role during fertilization and embryo development. A universal characteristic of oviductin is its association with the zona pellucida of ovulated oocytes, and several functional studies indicate an influence of this glycoprotein on reproductive events in mammals. The objective of this study was to produce recombinant feline oviductin in prokaryotic as well as eukaryotic cells as a prerequisite for analysing its influence on IVF in the domestic cat. Oviductin sequence was cloned into pET21b vector and transformed to E. coli to produce a recombinant His‐tagged, non‐glycosylated protein. Oviductin was isolated from E. coli lysate using anion exchange chromatography followed by immobilized metal ion affinity chromatography (IMAC). Western blot analysis of affinity purified fractions resulted in one clear band corresponding to the expected size of ~67 kDa. The corresponding band of a coomassie‐stained gel was analysed by mass spectrometry. Oviductin was identified with over 50 peptides covering 72% of the whole protein sequence. To obtain a glycosylated form of oviductin, eukaryotic cells were stable transfected with pSecTag/HygroA vector containing the oviductin gene sequence. The recombinant His‐tagged protein was harvested from a serum‐ and protein‐free cell culture medium. Mass spectrometry analyses of protein bands obtained after separation of the medium by SDS‐PAGE detected oviductin peptides in protein bands of ~70, ~85 and ~170 kDa. With prokaryotic as well as eukaryotic produced recombinant feline oviductin, we are now able to use the protein for further functional IVF studies in the domestic cat.
Clinical and Molecular Characteristics of Pyometra in Female Dogs
Authors: Hagman R
Published: December 2012

Contents

Pyometra is a common disease of female dogs. In Sweden, where approximately 90% of the dog population is intact (not neutered), nearly 25% of all female dogs are diagnosed with the disease before 10 years of age. In certain high‐risk breeds, this risk of developing pyometra exceeds 50%. Various clinical signs associated with the genital tract as well as with systemic disease are present in dogs with pyometra. A frequent and serious consequence of the uterine infection is endotoxaemia and progression into the systemic inflammatory response syndrome (SIRS), or sepsis, and the disease is then regarded as a medical emergency. Acute phase proteins and inflammatory markers associated with SIRS and with the outcome as measured by length of hospitalization have been identified in blood samples. Recently, the inflammatory response in infected uterine tissue during pyometra has been more closely explored. The expression of many genes associated with chemokines, cytokines, inflammatory cell extravasation, anti‐bacterial action, the complement system and innate immune responses and also a large panel of proteases are upregulated in the uterine tissue in pyometra. Products of certain upregulated genes may be detected systemically and used for diagnostic or prognostic purposes provided that tests are developed in the future. More knowledge of the complex local and systemic inflammatory response in pyometra may allow identification of novel disease biomarkers or future targets for treatment. In this article, clinical as well as molecular characteristics of the disease are reviewed.
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

Contents

Early 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

Contents

The 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

Contents

Pyometra 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

Contents

Somatic 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

Contents

In 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

Contents

Pre‐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

Contents

Artificial 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

Contents

Oxidative 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

Contents

Cryosurvival 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

Contents

Over 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

Contents

The 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

Contents

Molecular 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

Contents

Cryopreservation 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

Contents

Progesterone 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

Contents

Embryonic 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

Contents

The 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.
Use of Fluorescent Stainings and Flow Cytometry for Canine Semen Assessment
Authors: Niżański W , Partyka A , Rijsselaere T
Published: December 2012

Contents

Over 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

Contents

The 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

Contents

The 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

Contents

The 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

Contents

This 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

Contents

Our 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

Contents

In 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

Contents

Reproductive 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

Contents

The 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

Contents

In 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

Contents

The 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.