- Dose rates of antimicrobial substances in boar semen preservation—time
to establish new protocols
- Authors: M Schulze; M Grobbel, A Riesenbeck, S Brüning, J Schaefer, M Jung, R Grossfeld
Abstract: To achieve a standardized number of spermatozoa in the final AI dose, varying amounts of extender fluid with a fixed concentration of antimicrobial substances are currently added to boar ejaculates. This practice ignores the different degrees of dilution of the antimicrobials in the end product. In calculating the final concentration of gentamicin in AI doses from 27,538 processed boar ejaculates, we demonstrated varying gentamicin concentrations in the resultant extended boar semen samples. The median concentration was 220.37 mg/L. In 25 of the samples (0.09%), the gentamicin concentration fell below 5 mg/L, which is close to or below the epidemiological cut-off value for many bacteria. We calculated the minimum inhibitory concentration of gentamicin for bacteria isolated from raw and extended ejaculates. Five of the isolates from extended ejaculates exceeded the maximum test concentration of 512 mg/L. As a result, we are presenting an alternative method of boar semen preservation whereby a particular combination of gentamicin concentrate and antibiotic-free extender is incorporated that standardizes the antibiotic concentration in the diluted semen. The addition of standardized antibiotic concentrations did not negatively affect sperm quality when compared to the use of ready-to-use extenders. In conclusion, an end volume-based and standardized addition of gentamicin to boar ejaculates can be a helpful alternative to prevent insufficient dosage of antibiotics in liquid preserved boar semen without affecting semen quality.
- Effect of sperm pretreatment with glutathione and membrane destabilizing
agents lysolecithin and Triton X-100, on the efficiency of bovine
intracytoplasmic sperm injection
- Authors: F Zambrano; L Aguila, ME Arias, R Sanchez, R Felmer
Abstract: Intracytoplasmic sperm injection (ICSI) is an assisted reproduction tool with several applications. Its effectiveness in bovines is lower than that in other species, mainly because of difficulties in the decondensation of the sperm nucleus after injection, and the presence of the acrosome and the plasma membrane which remain intact in this procedure. In this study, we assessed the effect of lysolecithin (LL) and Triton X-100 (TX), in combination with glutathione (GSH) as sperm pretreatments prior to ICSI. The GSH-LL and GSH-TX groups showed 0% of spermatozoa with intact membrane (SYBR 14+/PI), in comparison with the control (63.3%) and GSH (65.7%) groups. The proportions of spermatozoa with damaged acrosome membrane in the GSH-LL, GSH-TX, GSH and control groups were 46%, 35.9%, 10.5% and 7.5%, respectively. Sperm chromatin decondensation analysis showed that the groups incubated for 3 hr with GSH presented greater decondensation (p
- Epigallocatechin-3-gallate (EGCG) and green tea polyphenols do not improve
stallion semen parameters during cooling at 4°C
- Authors: D Bucci; M Spinaci, B Mislei, B Gadani, G Rizzato, CC Love, C Tamanini, G Galeati, G Mari
Abstract: Stallion semen storage for artificial insemination is mainly based on liquid cooled storage. In many stallions this technique maintains sperm quality for an extended period of time (24–72 hr) at 7°C. While this technique is commonly used in the horse industry, there can be a decline in fertility in some stallions, due to an inability of their sperm to tolerate the cool storage process. The aim of the present work was to evaluate the effect of two natural antioxidants (epigallocatechin-3-gallate (EGCG) at 20, 60 and 120 μm and green tea polyphenols, and p at .001, .01 and .1 mg/ml) on some sperm parameters (sperm motility, viability/acrosome integrity and DNA quality) in extended semen immediately after its collection (T0) and after 2, 6, 24 and 48 hr of cool storage. Two ejaculates from three trotter stallions were analysed after 48 hr of storage at 4°C. No beneficial effect on the analysed parameters was observed: the two antioxidants were not able to improve sperm quality after 48 hr of storage. These results are in agreement with previous findings on the effect of different antioxidants reported by other researches, who have demonstrated that stallion semen keeps good antioxidant capacity after dilution for 24 hr. In conclusion, the positive effect exerted by antioxidant molecules in other species is not confirmed in the equine one.
- Lipid and protein oxidation levels in spermatozoa and seminal plasma of
Asian Elephants (Elephas maximus) and their relationship with semen
- Authors: S Satitmanwiwat; K Promthep, K Buranaamnuay, S Mahasawangkul, K Saikhun
Abstract: Peroxidation damage to spermatozoa and seminal plasma has an important role in sperm quality. Thus, the objective of this study was to determine the levels of lipid and protein oxidation in spermatozoa and seminal plasma of Asian elephants (Elephas maximus) with varying percentage of progressive motility. Lipid and protein oxidation was measured by the thiobarbituric acid-reactive species (TBARS) assay and the 2, 4-dinitrophenylhydrazine (DNPH) carbonyl groups assay, respectively. Fresh semen samples were collected from Asian elephants and classified according to the percentage of motile spermatozoa into good (>60%) and poor (≤20%) motility. Results revealed that seminal plasma malondialdehyde (MDA) and seminal plasma protein carbonyls (PCs) were significantly higher in poor motility than in good motility (p
- Effect of mitochondrial uncoupling and glycolysis inhibition on ram sperm
- Authors: JDA Losano; DSR Angrimani, A Dalmazzo, BR Rui, MM Brito, CM Mendes, GKV Kawai, CI Vannucchi, MEOA Assumpção, VH Barnabe, M Nichi
Abstract: Studies have demonstrated the importance of mitochondria to sperm functionality, as the main source of ATP for cellular homoeostasis and motility. However, the role of mitochondria on sperm metabolism is still controversial. Studies indicate that, for some species, glycolysis may be the main mechanism for sperm energy production. For ram sperm, such pathway is not clear. Thus, we evaluated ram sperm in response to mitochondrial uncoupling and glycolysis inhibition aiming to assess the importance of each pathway for sperm functionality. Statistical analysis was performed by the SAS System for Windows, using the General Linear Model Procedure. Data were tested for residue normality and variance homogeneity. A p
- Short communication: Progressive motility of frozen–thawed canine semen
is highest five minutes after thawing
- Authors: S Karger; B Geiser, M Grau, W Heuwieser, SP Arlt
Abstract: Progressive motility is usually estimated by visual inspection using a light contrast microscope at X 100 immediately after semen collection or immediately after thawing frozen semen. Standard operating procedures have never been established for this test. The objective of this experiment was to examine time-dependent changes of motility after thawing cryopreserved canine semen. Semen of 35 dogs was collected, and volume, concentration, progressive motility, morphology, membrane integrity and HOS test were evaluated. For cryopreservation, CaniPRO® Freeze A&B was used. Semen was thawed and diluted using CaniPRO® culture medium. After thawing, semen was evaluated as before. In addition, every sample was evaluated for progressively motile sperm cells 0, 5, 20 and 60 min after thawing. Progressive semen motility was significantly highest five minutes after thawing.
- Issue Information
- Pages: 1 - 2
- Pages: 179 - 179
- Prolactin and growth hormone immunoactivity in canine mammary adenomas and
- Authors: ER Bohrer; CV Löhr, MA Kutzler
Abstract: It is now widely accepted in human medicine that prolactin (PRL) and growth hormone (GH) function in the mammary gland in an autocrine and paracrine manner in tumour formation. The aim of this study was to compare PRL and GH immunoactivity in canine mammary tumours submitted for histopathologic evaluation. Formalin-fixed specimens from spontaneously occurring mammary adenomas and adenocarcinomas from 24 female client-owned dogs were used. Information pertaining to the reproductive status of the patient at the time of mammary tumour diagnosis was obtained from each of the submitting veterinarians. Tissues were paraffin-embedded and sectioned (5 μm) onto charged slides. All slides were deparaffinized and rehydrated. Endogenous peroxidase activity was inactivated with 3% H2O2, and non-specific binding was blocked. Polyclonal rabbit antihuman PRL (DAKO A0569) and GH antibody (DAKO A0570) were applied at a 1:250 and 1:200 dilutions, respectively. A universal rabbit negative control (DAKO N1699) was used. Slides were then reacted with anti-rabbit horseradish peroxidase followed by Nova Red Peroxidase substrate. Slides were counter-stained with haematoxylin, dehydrated and mounted. Tumour type and reproductive status at time of tumour diagnosis were compared individually between tumours that were negative or positive for PRL and GH using a two-tailed analysis of variance. Significance was defined as p
- Regulation of diapause in carnivores
- Authors: JC Fenelon; PL Lefèvre, A Banerjee, BD Murphy
Abstract: Embryonic diapause is an evolutionary strategy to ensure that offspring are born when maternal and environmental conditions are optimal for survival. In many species of carnivores, obligate embryonic diapause occurs in every gestation. In mustelids, the regulation of diapause and reactivation is influenced by photoperiod, which then acts to regulate the secretion of pituitary prolactin. Prolactin in turn regulates ovarian steroid function. Reciprocal embryo transplant studies indicate that this state of embryonic arrest is conferred by uterine conditions and is presumed to be due to a lack of specific factors necessary for continued development. Studies of global gene expression in the mink (Neovison vison) revealed reduced expression of a cluster of genes that regulate the abundance of polyamines in the uterus during diapause, including the rate-limiting enzyme in polyamine production, ornithine decarboxylase (ODC). In addition, in this species, in vivo inhibition of the conversion of ornithine to the polyamine, putrescine, induces a reversible arrest in embryonic development and an arrest in both trophoblast and inner cell mass proliferation in vitro. Putrescine, at 0.5, 2 and 1,000 μM concentrations induced reactivation of mink embryos in culture, indicated by an increase in embryo volume, observed within five days. Further, prolactin induces ODC1 expression in the uterus, thereby regulating uterine polyamine levels. These results indicate that pituitary prolactin acts on ovarian and uterine targets to terminate embryonic diapause. In summary, our findings suggest that the polyamines, with synthesis under the control of pituitary prolactin, are the uterine factor whose absence is responsible for embryonic diapause in mustelid carnivores.
- Long-term effects of GnRH agonists on fertility and behaviour
- Authors: S Goericke-Pesch
Abstract: This review aimed to summarize the present knowledge about the effects of GnRH agonist slow-release implants (GnRH A-SRI) on fertility and behaviour in male and female dogs and cats with special focus on deslorelin.Following an initial stimulation of gonadotropin and testosterone secretion possibly associated with an improved semen quality, GnRH A-SRI induce long-term depression of fertility in male dogs and cats with, however, a large individual variation in onset and duration of efficacy especially in cats. The GnRH A-SRI furthermore interfere with testosterone-dependent/affected behaviour; a significant positive effect in reducing sexual behaviour and libido, hypersexuality, intermale dominance and excessive territorial urine marking has been described. Rates of improvement of the respective behaviour are comparable to those after surgical castration, making GnRH A-SRI a valuable option to predict castration-related effects on behaviour and to identify animals where surgical castration will not be beneficial. No effect has been seen in reducing aggression towards humans indicating the need for behavioural therapy to control this problem. Effects on spermatogenesis, steroidogenesis and behaviour have by now been shown to be fully reversible. Knowledge in females is more limited, and particularly, the initial induction of a possibly fertile oestrus and individual variation in duration of efficacy remain problems in bitches and queens treated for suppression of fertility. However, long-term suppression of oestrous cycle and fertility seems to be possible with induced effects shown to be reversible including restoration of normal fertility after the end of efficacy/GNRH A-SRI removal.
- Changes in acetylation of lysine 5 on histone H4 in canine oocytes
following in vitro maturation
- Authors: TF Motheo; DR Arnold, LC Padilha-Nakaghi, EA Pires-Buttler, AE Alves, M Apparicio, WRR Vicente, FL Lopes
Abstract: Post-translational modifications of histones, such as acetylation, are involved in regulating chromatin remodelling and gene expression. Proper in vitro maturation (IVM) of canine oocytes, for many reasons, is up to now inefficient. This study aimed to evaluate the post-translational histone H4 acetylation at lysine 5 (H4K5) in immature and post-IVM canine oocytes. Oocyte nuclear stage was assessed using Hoechst 33342 staining. Acetylation patterns were determined by indirect immunofluorescence staining of immature and post-IVM oocytes, using an antibody against the acetylated lysine 5 residue on histone 4 (H4K5ac). The experiment was repeated four times, with a total of 7–17 oocytes evaluated per stage. Immunofluorescence signal was quantified using the NIHimagej software. Data were expressed as a percentage of the average fluorescence intensity of the specific antibody over the intensity of DNA, as determined by Hoescht staining. H4K5ac displayed a significantly higher acetylated pattern in immature oocytes (0.97 ± 0.08) when compared to post-IVM oocytes at different nuclear stages. There was a decrease in the fluorescence level of the matured oocytes with the progression of meiosis (GVBD: 0.47 ± 0.06 and MI/MII: 0.35 ± 0.04). Similarly to other domestic species, we hypothesized that post-translational modification of histone acetylation takes place during meiosis of in vitro matured canine oocytes. However, it remains to be investigated whether these changes occur during in vivo maturation.
- Assessment of spermatozoa in fertile alpaca (Vicugna pacos) males: Study
of sperm head morphometry using a nonautomated digital method and sperm
morphology based on strict criteria
- Authors: D Evangelista-Vargas; S Evangelista-Vargas, M Valdivia, A Santiani
Abstract: Although computer-assisted systems for sperm morphometry and morphological analysis are important tools in the study of male fertility, their use in extensive systems in alpacas is limited by factors such as the expense of equipment and the high altitudes of the Andean region. The objectives of this study were to evaluate alpaca sperm head morphometry using a nonautomated digital method and determine the frequency of sperm abnormalities based on strict criteria for sperm morphology in fertile male alpacas. Ejaculates (n = 15) from seven alpacas were collected, and sperm smears stained with modified Papanicolaou were processed. For morphometric analysis, 3,000 sperm (200 cells/sample) images were captured at 400× magnification and Quick Photo MICRO 3.0 software was used for manual measurement of basic (sperm head length, width, perimeter and area) and derived variables (ellipticity, shape factor, elongation and regularity). For morphology assessment, smears were observed at 1000× magnification according to WHO and strict criteria. Average morphometric parameters were length 5.48 μm, width 2.99 μm, perimeter 13.62 μm, area 12.43 μm2, ellipticity 1.86, shape factor 1.20, elongation 0.29 and regularity 1.05. Significant between-individual and within-individual differences were found in morphometric parameters. Based on morphometric study, sperm heads were classified as elliptical or normal (49%), long (18%), short (2%), pyriform (12%), round (9%), large (6%) and small (4%). Morphological analysis found no additional sperm head defects in 49% of normal sperm obtained by morphometry, although a 4% incidence of neck/mid-piece defects and a 16% incidence of principal-piece defects were found. We conclude that sperm head morphometry assessment in fertile alpacas using a nonautomated digital method is feasible, and that defects in sperm heads constitute the main morphological alteration (>50% of the sperm population), based on WHO and strict criteria.
- Coxiella burnetii: Serological reactions and bacterial shedding in
primiparous dairy cows in an endemically infected herd—impact on milk
yield and fertility
- Authors: M Freick; H Enbergs, J Walraph, R Diller, J Weber, A Konrath
Abstract: Coxiella burnetii (C. burnetii) is the causative agent of Q fever both in humans and animals. The objectives of this study were to investigate seropositivity and bacterial shedding in heifers and primiparous cows in an endemically infected herd and to assess the effects on post-partum diseases, fertility and milk production. At the age of 9 months, 96 Holstein heifers were included. Sampling was performed reproduction-orientated: at the beginning of the study, at detection of first pregnancy, 3 weeks before expected calving date (blood serum), at parturition and after 21, 42, 100 and 150 days in milk (DIM) (blood serum, vaginal swabs and milk). Serum samples were investigated by a commercial ELISA for the presence of specific antibodies and vaginal swabs and milk samples by PCR to detect C. burnetii DNA. Individual animal data (calving ease, stillbirth, retained foetal membranes, puerperal metritis, endometritis after 42 DIM, presence of corpus luteum after 42 DIM, interval calving-first service, interval calving-conception, number of inseminations until 150 DIM, proportion of pregnant cows until 100 and 150 DIM, proportion of pregnant cows after first service and data of the dairy herd improvement test) were documented. All heifers were seronegative at the age of 9 months and 3 weeks before the expected calving date. Subsequently, the proportion of seropositive animals and the antibody score increased significantly towards 42 and 100 DIM, respectively. Vaginal C. burnetii shedding was highest at parturition (30.9%), while the most positive milk samples were detected after 100 DIM (15.3%). Coxiella burnetii seropositivity and shedding had no impact on parameters of reproduction. However, milk fat yield was declined in puerperal vaginal shedders and cows which seroconverted during their first 42 DIM, respectively.
- Oxytocin receptors in dioestrous and anoestrous canine uteri
- Authors: TM Tamminen; L Sahlin, B Masironi, J Taponen, O Laitinen-Vapaavuori, T Katila
Abstract: The aim of the study was to localize oxytocin receptors (OTR) and measure mRNA expression of OTR in the canine uterus with and without the influence of progesterone. Uterine samples were taken from nine anoestrous and eight dioestrous bitches during ovariohysterectomy. Histological changes were evaluated in haematoxylin and eosin (HE)-stained samples. Purified polyclonal antibody for OTR was used in immunohistochemistry to localize receptors in uterine layers. Relative mRNA concentration of OTR was evaluated with real-time PCR from full-thickness uterine samples taken from the middle horn and the body. Myometrial smooth muscle cells, endometrial luminal epithelium (LE) and deep and superficial glandular epithelium were positively stained for oxytocin receptors in non-pregnant animals. No significant difference in staining intensity was detected between uterine middle horn and body. However, the staining intensity of LE was significantly higher in dioestrous than in anoestrous uteri (p
- The onset of puberty in Cameroon Dwarf goats kept as pets in northwestern
- Authors: D Đuričić; S Vince, H Valpotić, I Žura Žaja, R Turk, M Lojkić, I Getz, V Berta, M Samardžija
Abstract: The aim of this study was to determine onset of puberty in Cameroon Dwarf goats (CDGs) kept as pets in northwestern Croatia by determining progesterone (PGS) and insulin-like growth factor-I (IGF-I) levels in the blood by RIA methods. The first cyclic ovarian activity was estimated according to hormone profiles as determined in CDG in a moderate climate environment. Sixteen female CDG kids were kept in stables with access to pasture which provided space for exercise. The goat kids born in winter (December–January–February), spring (March–April–May), summer (June–July–August) and autumn (September–October–November) were assigned into four groups according to the season of the year of birth (n = 4 in each group). At 75 days of age at the initiation of the study, they weighed between 3.2 and 5.1 kg (4.24 ± 0.53 kg). The onset of ovulatory activity was determined by PGS and IGF-I serum concentrations every 10 days starting from 75 days to 155 days of age. The onset of puberty in CDG kids occurred on average at 141.15 ± 2.66 days of age, but varied depending on the season of birth. All CDG kids born during summer were in heat by 155 days of age. In the other groups (kids born in autumn, winter or spring), one goat in each group was not in heat. Changes in blood serum PGS and IGF-I concentrations during prepubertal and pubertal periods could aid in the evaluation of reproductive status and determination of the onset of puberty in CDG during all seasons of the year in a moderate climate region.
- Efficiency of uterine fluid cytology in the diagnosis of subclinical
endometritis in the water buffalo (Bubalus bubalis)
- Authors: SC Gahlot; S Kumar, A Kumaresan, S Chand, RK Baithalu, S Lathika, TK Patbandha, SS Lathwal, TK Mohanty
Abstract: This study compared endometrial cytology vis-a-vis uterine fluid cytology for assessment of uterine health in clinically normal and subclinical endometritis (SE)-affected buffaloes. Uterine fluid samples and endometrial samples were collected from the buffaloes (n = 38) at oestrus using blue sheath and cytobrush, respectively. The smears were stained with Field stain for 3 minutes, and a minimum of 400 cells were counted in each smear for determination of the percentage of polymorphonuclear (PMN) leucocyte. The incidence of subclinical endometritis, based on the cytobrush cytology, was 23.08%. The correlation between cytobrush cytology with uterine fluid cytology was positive and significant (r = .37; p = .02). The ratio of PMN leucocyte in cytobrush cytology to uterine fluid cytology was 1:2.4. ROC analysis revealed that the threshold value of 6.16% PMN leucocyte in uterine fluid cytology showed a diagnostic sensitivity and specificity of 100% in differentiating normal from SE-affected buffaloes. In conclusion, collection of uterine fluid was easier compared to collection of endometrial samples using cytobrush and the percentage of PMN leucocyte in uterine fluid cytology can be used as a tool for diagnosis of subclinical endometritis in buffaloes.
- Antisperm antibodies in repeat-breeding cows: Frequency, detection and
validation of threshold levels employing sperm immobilization, sperm
agglutination and immunoperoxidase assay
- Authors: SK Srivastava; S Shinde, SK Singh, S Mehrotra, MR Verma, AK Singh, S Nandi, N Srivastava, SK Singh, TK Goswami, SK Bhure, H Kumar, SK Ghosh
Abstract: Antisperm antibodies have been found in repeat-breeding(RB) cows, and those causing agglutination and/or immobilization of sperm are considered to be closely related to unexplained infertility. However, a standard protocol for identifying antisperm antibodies (ASA) in cattle is not validated. Therefore, an investigation was undertaken to evaluate sperm immobilization (SIT), sperm agglutination (SAT) and immunoperoxidase (IPT)assays for detection of ASA in serum and their respective threshold levels for confirmation. Animals (heifers, normally breeding, repeat-breeding and pregnant animals) that were free from IBR, brucellosis and uterine infections (screened by clinical examination) were included in the study. Sperm agglutinating, sperm immobilizing and antisperm antibodies evaluated by respective assay were significantly higher (p
- Extracellular matrix in epitheliochorial, endotheliochorial and
haemochorial placentation and its potential application for regenerative
- Authors: ARA Anunciação; AM Mess, D Orechio, BA Aguiar, PO Favaron, MA Miglino
Abstract: A placenta is defined as structural approximation of maternal and foetal tissues to perform physiological exchange. Associated processes of differentiation and the establishment of its cells take place within the extracellular matrix (ECM) that provides a rich environment of collagens, fibronectins, cytokines and other components. Placental ECM is promising for tissue regeneration purposes, because it has immune tolerance capacities that may cause only minimal rejections of transplants with immunological differences between donor and recipient. However, specific characteristics of ECM during evolution of the structurally very diverse mammalian placenta are not yet revealed. We here address the major aspects of placental types, that is non-invasive (epitheliochorial), medium (endotheliochorial)-to-high (haemochorial) invasive nature of the interhemal barrier between the foetal and maternal blood system as well as their main components of ECM with special reference to species that are commonly used as animal models for human placentation and in the potential applications for regenerative medicine.
- A new device to inseminate cows at the base of the uterine horns
- Authors: O García-Peña; R Rangel-Santos, R Rodríguez-De Lara, CA Apodaca-Sarabia, E Maldonado-Simán
Abstract: A new device (Chapingo device) to deposit semen at the base of the uterine horns of cattle was developed at Universidad Autonoma Chapingo, Mexico. Nine Holstein heifers were inseminated by transvaginal laparoscopy, using a laparoscope for cattle and the Chapingo device. A dose of sexed semen (2.1 × 106 spermatozoa) was deposited at the base of the uterine horn ipsilateral to the ovary where the preovulatory follicle was identified. Insemination was achieved in all the heifers, taking on average 13.7 ± 3.1 min per animal. In all cases, it was possible to see both ovaries, the base of the uterine horns and the oviducts. After the procedure, none of the heifers showed any type of complications such as haemorrhage, adhesions or trauma. On days 21 and 22 after insemination, four of the nine heifers (44.4%) returned into oestrus; on day 30 after insemination, one heifer was found to be pregnant by ultrasound. The results show the feasibility of generating pregnancies by transvaginal laparoscopy in heifers inseminated with sexed semen.
- Isolation, proliferation and characterization of endometrial canine stem
- Authors: V De Cesaris; S Grolli, C Bresciani, V Conti, G Basini, E Parmigiani, E Bigliardi
Abstract: In the last decade, progenitor cells isolated from dissociated endometrial tissue have been the subject of many studies in several animal species. Recently, endometrial cells showing characteristics of mesenchymal stem cells (MSC) have been demonstrated in human, pig and cow uterine tissue samples. The aim of this study was the isolation and characterization of stromal cells from the endometrium of healthy bitches, a tissue that after elective surgery is routinely discarded. Multipotent stromal cells could be isolated from all bitches enrolled in the study (n = 7). The multipotency of cells was demonstrated by their capacity to differentiate into adipocytic, osteocytic and chondrocytic lineages. Clonogenicity and cell proliferation ability were also tested. Furthermore, gene expression analysis by RT-PCR was used to compare the expression of a set of genes (CD44, CD29, CD34, CD45, CD90, CD13, CD133, CD73, CD31 CD105, Oct4) with adipose tissue-derived MSC. Stromal cells isolated from uterine endometrium showed similar morphology, ability of subculture and plasticity, and also expressed a panel of genes comparable with adipose tissue-derived MSC. These data suggest that endometrial stromal cells fulfil the basic criteria proposed by the “Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy” for the identification of mesenchymal stem cells. Although endometrial mesenchymal stem cells (EnMSC) showed a lower replicative ability in comparison with adipose tissue-derived MSC, they could be considered a cell therapeutic agent alternative to adipose tissue or bone marrow-derived MSC in dog.
- Bovine ovarian stem cells differentiate into germ cells and oocyte-like
structures after culture in vitro
- Authors: GB Souza; JJN Costa, EV Cunha, JRS Passos, RP Ribeiro, MVA Saraiva, R Hurk, JRV Silva
Abstract: Stem cells have been isolated from ovaries, and their ability to differentiate into oocytes in vitro has been demonstrated for mice and human, but not for bovine species. The aims of this study were to isolate germline stem cells from bovine ovaries and to evaluate the effects of bone morphogenetic proteins (BMPs) 2 and 4, and follicular fluid on the differentiation of these stem cells into oocyte-like structures. The ovarian stem cells were isolated and cultured in α-MEM+ supplemented with BMP2, BMP4 or follicular fluid. On days 0 and 14, cells were evaluated for their morphological appearance, viability, expression of alkaline phosphatase and for markers of germ cell formation (VASA and DAZL) and oocyte development (GDF9, ZPA and SCP3) by qPCR. Levels of mRNA were analysed using ANOVA and Bonferroni test (p
- Environmental and maternal factors associated with gross placental
morphology in dairy cattle
- Authors: MM Kamal; M Van Eetvelde, L Vandaele, G Opsomer
Abstract: This article reports on a study of gross placental morphology of 282 expelled placentas from 89 primi- and 193 multiparous Holstein dams immediately after calving and examines associations with environmental factors such as typical herd features and season of calving, and maternal factors such as age at calving, level of milk yield at conception and cumulative amount of milk produced during gestation. The highest correlation between calf measurements and placental characteristics was found between the weight of the calf and the total cotyledonary surface (r = .643; p
- Evaluation of epididymis storage temperature and cryopreservation
conditions for improved mitochondrial membrane potential, membrane
integrity, sperm motility and in vitro fertilization in bovine epididymal
- Authors: M Nichi; T Rijsselaere, JDA Losano, DSR Angrimani, GKV Kawai, IGF Goovaerts, A Van Soom, VH Barnabe, JBP De Clercq, PEJ Bols
Abstract: The maintaining of the epididymis at lower temperatures during storage and transport improves sperm quality. Our study aimed to test whether epididymis storage temperature (post-mortem) and sperm cryopreservation affect sperm kinetics, membrane integrity, mitochondrial potential and fertility capacity. Thirty-six epididymides were collected from 18 bulls after slaughter and divided into two groups: at 4 or 34°C for 2–3 hr. The sperm was collected from the epididymis cauda. The evaluation consisted of computer-assisted sperm analysis (CASA), SYBR14/PI/JC1 to evaluate membrane integrity, mitochondrial membrane potential (MMP) and measurement of lipid peroxidation (TBARS). The sperm was then frozen using an automatic device. After thawing, sperm samples were evaluated by the same variables and further in vitro fertilization rates. Cryopreservation negatively affected sperm motility in samples stored at 4 and 34°C. Nevertheless, the 4°C samples yielded higher rates of blastocyst formation. Pre-freeze sperm motility, progressive motility and velocity were higher in sperm from epididymis stored at 4°C while post-thaw sperm motility, progressive motility and velocity remained the same among samples from epididymis stored at 4 or 34°C. However, with regard to the kinetic patterns, samples collected from epididymis stored at 34°C had lower values when compared to those stored at 4°C prior the cryopreservation process. Our results indicate that epididymis handling conditions after cryopreservation may affect sperm quality after thawing, especially due to compromised MMP in sperm collected from epididymis stored at higher temperatures.
- First case of sterility associated with sex chromosomal abnormalities in a
- Authors: J Dorado; G Anaya, M Bugno-Poniewierska, A Molina, A Mendez-Sanchez, I Ortiz, M Moreno-Millán, M Hidalgo, P Peral García, S Demyda-Peyrás
Abstract: Chromosomal abnormalities are one of the main causes of genetic infertility in horses. Currently, their detection rate is rising due to the use of new diagnostic tools employing molecular markers linked to the sex chromosome pair. Despite genetic similarities, there are no previous reports of sterility associated with chromosomal abnormalities in the domestic donkey (Equus asinus). Hereby, we determined the presence of a chromosomal mosaicism in a female donkey with reproductive problems using molecular methodologies developed for horses. A two-and-a-half-year-old jenny characterized by morphological abnormalities of the reproductive tract was cytogenetically analysed using conventional and fluorescent techniques and a group of microsatellite markers (short tandem repeat, STR). At the same time, five ultrasound measures of the reproductive tract were taken and compared with eight contemporary jennies of the same breed. After slaughter, morphological examinations showed that the case study had a blind vaginal vestibule defining an empty pouch that covered the entrance of the cervical os. Histopathological studies demonstrated that this abnormal structure was compatible with a remnant hymen. Molecular markers, STR and fluorescent in situ hybridization determinations revealed that the animal was a 62, XX/61,X mosaic and, therefore, the first case of chromosomal abnormalities in the sex pair reported in donkeys.
- The effect of cumulus cells on domestic cat (Felis catus) oocytes during
in vitro maturation and fertilization
- Authors: N Sowińska; K Frankowska, A Filipczyk, A Adamaszek, K Nalik, K Fic, A Pietsch-Fulbiszewska
Abstract: The aim of this study was to evaluate the effect of co-culture of denuded oocytes with cumulus cells (CC) or cumulus–oocyte complexes (COCs) on in vitro maturation (IVM) and in vitro fertilization (IVF). Immature oocytes were collected from ovaries of domestic cats following a routine ovariectomy. Oocytes were matured in vitro for 24 hr within four groups: (i) denuded oocytes (DO), (ii) DO co-cultured with CC, (iii) DO co-cultured with COC and (iv) COC as a control group. In further experiments, COCs were matured in vitro for 24 hr, and then, oocytes were randomly divided into four groups as previously described and fertilized in vitro. Embryos were cultured for up to 7 days. At the end of each experiment, oocytes/embryos were stained with Hoechst 33342 solution and observed under an inverted fluorescence microscope. The results of oocyte maturation showed that their meiotic competence decreased significantly in all experimental groups, compared to the control group. The maturation rates were approximately 45%, 24%, 43% and 76% in experiment 1, and 21%, 14%, 33% and 50% in experiment 2 in groups (i), (ii), (iii) and (iv), respectively. Examination of in vitro fertilization revealed that embryos developed up to the morula stage in all experimental groups. DO and oocytes cultured with COC during fertilization showed a lower cleavage rate—36% and 25% as opposed to those co-cultured with loose CC and the control group—43% and 42%, respectively. Results of this study indicate that cumulus cells connected with an oocyte into a cumulus—oocyte complex are irreplaceable for the maturation of domestic cat oocyte, but that the addition of loose CC may be beneficial for IVF.
- Temporal changes in serum luteinizing hormone following ovariohysterectomy
and gonadotropin-releasing hormone vaccination in domestic cats
- Authors: HL Bateman; LM Vansandt, J Newsom, WF Swanson
Abstract: Measurement of circulating luteinizing hormone (LH) concentrations in cats and temporal changes following ovariohysterectomy (OHE) or possibly GnRH vaccination may be informative for assessing their fertility, contraception or sterilization status. In this study, serum LH concentrations were measured in domestic cats (n = 6) immediately prior to and up to 120 days post-OHE. Basal LH concentrations of females previously subjected to OHE (n = 4; ~1.5 years post-OHE) were compared pre- and post-vaccination with a GnRH immunocontraceptive, and to LH concentrations in intact females. Basal serum LH concentrations (2.67 ± 0.43 ng/ml; mean ± SEM) in intact females increased (p
- Canine and feline colostrum
- Authors: S Chastant-Maillard; C Aggouni, A Albaret, A Fournier, H Mila
Abstract: Puppy and kitten survival over the first weeks is particularly dependent on colostrum, a specific secretion of the mammary gland produced during the first 2 days post-partum. Colostrum is a source of nutrients and immunoglobulins. It also contributes to the digestive tract maturation. Colostrum differentiates from milk mainly based on its concentration in immunoglobulins G: 20–30 g/L in dog colostrum, 40–50 g/L in cats’ vs
- Distribution of follicles in canine ovarian tissues and
xenotransplantation of cryopreserved ovarian tissues with even
distribution of follicles
- Authors: I Wakasa; M Hayashi, Y Abe, H Suzuki
Abstract: Ovarian follicles are not homogeneously distributed within the ovarian cortex in several species of mammals. Yet to maximize the reproducibility of experimental results of ovarian transplantation, it is essential to assess the degree of density and distribution of follicles in ovarian tissues before their transplantation. In this study, the ovarian cortex from 13 immature bitches (ten purebred and three mongrels) was sectioned into 1.0- to 1.5-mm3 cubes, those were fixed, sectioned and stained with haematoxylin and eosin. To evaluate the density and distribution of follicles, the mean number of all stages of follicles per square millimetre was calculated after observation under a microscope. The distribution of follicles was considered even when the variance value was lower than 10 or between 10 and 16, with an absolute value of distortion inferior to 1. The mean number of follicles ranged from 3.24 to 28.34/mm2 in 25 ovaries from 13 bitches examined. The variance and distortion ranged from 0.35 to 119.64 and −1.87 to 4.40, respectively. The distribution of follicles within the ovarian cortex was judged uneven in 12 of 25 ovaries. These results indicated that follicles were not homogeneously distributed within the ovarian cortex in a large proportion of ovaries. In addition, cryopreserved ovarian fragments with even distribution of follicles were transplanted to NSG mice with or without 400 U/kg of disialylated erythropoietin (asialo EPO). After removing both sides of ovary, a piece of ovarian fragment was placed under the kidney capsule in both sides of kidney. At 4 weeks after transplantation, the fragments were recovered from the mice and the number of primordial, primary, secondary and antral follicles was counted. Total number of follicles and survival rates of follicles in transplanted fragments with asialo EPO were higher than without asialo EPO in four bitches examined. These findings suggest that asialo EPO might be effective on the follicular survival of canine ovarian tissues after xenotransplantation. Knowing the degree of density and distribution of follicles in ovarian tissues before transplantation is expected to contribute to the precise interpretation of results after transplantation of the ovarian tissues.
- New approaches to non-surgical sterilization for dogs and cats:
Opportunities and challenges
- Authors: Linda Rhodes
Abstract: Over the last 40 years, researchers have explored methods to non-surgically suppress fertility in animals. Immunocontraception has been used to control wildlife populations but does not confer long-term immunity. The gonadotropin-releasing hormone (GnRH) agonist deslorelin, formulated as an implant to provide 6-month to 1-year suppression of fertility in male dogs, is available commercially in some countries. Neither of these approaches provide permanent sterility. A single-dose, permanent treatment would be a valuable tool in dog and cat population control. The Michelson Prize and Grants (MPG) programme was initiated “to eliminate shelter euthanasia of healthy, adoptable companion animals and reduce populations of feral and free-roaming cats and dogs” offering a $25 million US prize for a non-surgical sterilant that is effective as a single treatment in both male and female dogs and cats. Michelson Prize and Grants programme has offered US $50 million in grant money for research and has attracted scientists worldwide. Approaches under study include gene therapy, small interfering RNA to inhibit reproductive targets and delivery of cytotoxins to pituitary gonadotrophs or GnRH producing neurons in the hypothalamus. Research in implant technology that could deliver compounds over an animal's lifetime is also underway. Details of funded grants and results to date can be found at: http://www.michelsonprizeandgrants.org/michelson-grants/research-findings. The next steps are translating the most promising research into products. The Alliance for Contraception of Cats and Dogs (ACC&D) is helping to research practical methods of marking sterilized animals to avoid costly retreatment and population modelling that will help guide field workers in use of resources for sterilization programmes.
- Identification of lactoferrin and glutamate receptor-interacting protein 1
in bovine cervical mucus: A putative marker for oestrous detection
- Authors: WY Lee; MH Park, KW Kim, H Song, KB Kim, CS Lee, NK Kim, JK Park, BC Yang, KB Oh, GS Im, HJ Chung
Abstract: Accurate detection of oestrus is important for artificial insemination. The aim of this study was to identify oestrous-specific bovine cervical mucus proteins that could be used to determine the optimal time for artificial insemination. Non-oestrous and controlled internal drug release (CIDR)-induced oestrous-stage mucus proteins were purified and subjected to surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, sodium dodecyl sulphate polyacrylamide gel electrophoresis and MALDI-TOF/TOF. Among differentially expressed proteins, lactoferrin (LF) and glutamate receptor-interacting protein 1 (GRIP1) showed a twofold increase during the CIDR-induced oestrous stage compared to the levels in non-oestrous stage in bovine cervical mucus. The RT-PCR, Western blotting and immunohistochemistry results showed that LF and GRIP1 expression was significantly increased during the oestrous stage in the uterus. This study demonstrated that bovine LF and GRIP1 exist during the oestrous stage, but not during the non-oestrous stage, suggesting that cervical mucus LF and GRIP1 are useful oestrous detection markers in cattle.
- A retrospective clinical study of endoscopic-assisted transcervical
insemination in the bitch with frozen–thawed dog semen
- Authors: SJ Mason
Abstract: Since the conclusion of data collation from previously published work, a further 352 inseminations using frozen–thawed dog semen by endoscopic-assisted transcervical insemination (EIU) have been performed by the author. Insemination was performed on the second day in which crenulation of the anterior vagina was detected in conjunction with a progesterone concentration of >10 ng/ml. All semen samples were analysed for total number of sperm, total motility and progressive motility using computer-assisted semen analysis (CASA). The insemination dose was based on the progressively motile normal spermatozoa (PMNS). Insemination was performed on all bitches as previously described using a ureterorenoscope. Additional extender was inseminated subsequent to the semen to expand and fill the uterus. The semen and additional extender were inseminated slowly over a period of 15–20 min. Pregnancy was determined by B-mode ultrasound equipped with a 7.5-MHz probe whilst standing and/or via the whelping rate. The number of sperm inseminated ranged from 9 × 106 PMNS to 519 × 106 PMNS, with progressive motility values ranging between 20% and 80%. The overall pregnancy rate was 68% (238/352). When stratified by PMNS, pregnancy rates were as follows: >150 × 106 PMNS - 76% (110/145), 100–150 × 106 - 68% (87/128) and 150 × 106 PMNS (p = .003) or 100–150 ×106 PMNS (p = .027) were inseminated compared to 150 × 106 PMNS to maximize pregnancy rate. These results indicate that one optimally timed EIU insemination results in similar pregnancy rates to previous publications of one optimally timed, or two or more non-optimally timed inseminations using the Norwegian catheter.
- Cryopreservation of feline oocytes by vitrification using commercial kits
and slush nitrogen technique
- Authors: L Fernandez-Gonzalez; K Jewgenow
Abstract: Assisted reproductive techniques are a valuable tool for conservation breeding of endangered species. Cryopreservation methods are the basis of gamete banks, supporting genetic diversity preservation. Unfortunately, cryopreservation of feline oocytes is still considered an experimental technique. The aim of this study was to compare two commercial kits, with our protocol for vitrification of cat oocytes (IZW), which comprises a three-step method with ethylene glycol, DMSO, fetal calf serum, trehalose and Ficoll PM-70. Furthermore, we applied slush nitrogen (SN2) for ultra-rapid freezing to improve survival rates. Cumulus–oocyte complexes were collected from domestic cat ovaries by slicing and vitrified at immature stage using Cryotop as storage device. Vit Kit® Freeze/Thaw (n = 89) showed the lowest maturation percentage obtained after warming (10.1%). A significant difference in maturation percentage of oocytes was found between Kitazato® kit (38.7%, n = 137) and IZW protocol (24.5%, n = 143). The cleavage after ICSI of warmed and matured oocytes (20.7% and 28.6%, respectively) and the morula percentage (18. 2% and 22.5%, respectively), however, did not reveal any significant difference between the two methods. Application of SN2 did not result in any improvement of oocytes’ cryopreservation. Maturation percentage of the oocytes vitrified by IZW method with SN2 (n = 144) decreased until 6.1%, without any cleavage after fertilization. For Kitazato® (n = 62), only 17.7% were able to undergo maturation and cleavage percentage dropped to 18.2%, not reaching morula stage. These data demonstrate that feline oocytes can be vitrified either by our IZW method or by commercial Kitazato® kit, but the use of SN2 is improving neither maturation nor cleavage percentages when combined with these procedures.
- Beneficial effect of extracellular adenosine 5′-triphosphate treatment
on the Indochinese leopard (Panthera pardus delacouri) sperm quality after
- Authors: P Thuwanut; W Tipkantha, B Siriaroonrat, P Comizzoli, K Chatdarong
Abstract: The Indochinese leopard (Panthera pardus delacouri) population, included in CITES Appendix I, has been declining for decades. Proper gamete preservation condition is critical for breeding programme management using artificial insemination or in vitro fertilization (IVF). The present study aimed at investigating the impact of post-thawing treatment of leopard semen with extracellular adenosine 5′-triphosphate (ATPe) on sperm quality (including morphological traits and ability to fertilize an oocyte). Semen from six adult male leopards was collected by electroejaculation (one ejaculation per cat). After the evaluation of the fresh sample quality, the semen was cryopreserved (10 × 106 cells per straw; two straws per cat). After thawing, the sperm sample from the first straw of each cat was divided into three aliquots: control (no ATPe), supplemented with 1.0 or 2.5 mM ATPe that were evaluated for sperm quality at 10, 30 min and 3 hr post-thawing. The sperm sample from the second straw, supplemented with 0, 1.0 or 2.5 mM ATPe for 30 min, was assessed for IVF with domestic cat oocytes. Sperm quality (all metrics) was negatively affected by the cryopreservation process (p ≤ .05). However, the percentage of sperm motility, level of progressive motility and percentage of plasma membrane integrity did not differ (p > .05) among post-thawing groups. The sperm mitochondrial membrane potential was enhanced (p ≤ .05) by ATPe treatment (1.0 and 2.5 mM; 10 min to 3 hr of incubation). Furthermore, incubation of ATPe (1.0 and 2.5 mM) for 30 min could promote sperm velocity patterns (curvilinear velocity; VCL and straight line velocity; VSL) (p ≤ .05). The percentage of pronuclear formation and cleaved embryos was increased (p ≤ .05) after 1.0 ATPe treatment (49.8 ± 2.8; 45.9 ± 1.5) compared to 0 mM (41.4 ± 3.3; 38.9 ± 0.5) whereas the number of sperm binding/oocyte did not significantly differ among groups. In summary, we suggest that ATPe activated the velocity of Indochinese leopard sperm motility that may lead to faster sperm/oocyte binding and sperm penetration (factors of successful embryo development).
- Effects of supplemental dietary vitamin C on quality of semen from Nile
tilapia (Oreochromis niloticus) breeders
- Authors: NLAF Sarmento; EFF Martins, DC Costa, WS Silva, CC Mattioli, MR Luz, RK Luz
Abstract: The objective of this study was to evaluate the effects of vitamin C on growth and quality of semen from Oreochromis niloticus breeders. One hundred and sixty males were fed with different levels of vitamin C (0, 261, 599 and 942 mg/kg diet). The higher weight values were recorded for 599 (166 g) and 942 (175 g) mg of vitamin C/kg diet. Sperm motility, vigour and concentration were higher with 599 and 942 mg of vitamin C/kg diet. The semen volume, gonadosomatic index and plasma protein data from the last week showed a direct relationship with increasing levels of vitamin C. No changes were observed in the hepatosomatic index and blood glucose. The haematocrit and erythrocyte showed higher values estimated by equations derived at 850 and 638 mg vitamin C/kg diet, respectively. The leucocytes were inversely proportional to the increasing levels of vitamin C. After 100 days of feeding, animals fed the diet containing 942 mg vitamin C/kg diet had higher sperm motility, linearity, curvilinear velocity, straight line velocity and average path velocity (p
- Monochorial diamniotic dizygotic twins in a German Shepherd Dog: A case
- Authors: C Urhausen; K Wolf, A Beineke, C Dierks, M Schmicke, A Einspanier, AR Günzel-Apel
Abstract: Case report: A 6.5-year-old clinically healthy German Shepherd Dog with regular oestrous cycles of 6 months was presented for pregnancy diagnosis on day 38 after ovulation (p.ov.). Ultrasonography revealed three individual placental sites in progressed resorption and two vital adequately developed foetuses sharing a joint placenta. On days 41 and 48 p.ov., sonographic signs indicated normal development of both foetuses, but on day 52 p.ov., both foetuses were found to be dead. A caesarean section was performed the same day. Examination of the removed uterus confirmed the diagnosis of a “twin” pregnancy with two foetuses sharing the same placental site but separate amniotic membranes. One foetus showed generalized oedema (anasarca). Bacterial culture of swabs taken from inside the placental cavity was negative. At histological examination of the uterus, no signs of inflammation were found. Serum relaxin concentrations (day 38, 41, 48 and 52. p.ov.) were consistent with those of bitches with normal pregnancies. Cytogenetic analysis of the two foetuses revealed dizygotic twins, one male and one female according to SRY-PCR. By genotyping 17 high-polymorphic canine microsatellites, it could be demonstrated that the two foetuses developed from two different oocytes.
- Engineering a gene silencing viral construct that targets the cat
hypothalamus to induce permanent sterility: An update
- Authors: GA Dissen; K Adachi, A Lomniczi, T Chatkupt, BL Davidson, H Nakai, SR Ojeda
Abstract: The intent of this contribution is to provide an update of the progress we have made towards developing a method/treatment to permanently sterilize cats. Our approach employs two complementary methodologies: RNA interference (RNAi) to silence genes involved in the central control of reproduction and a virus-based gene therapy system intended to deliver RNAi selectively to the hypothalamus (where these genes are expressed) via the systemic administration of modified viruses. We selected the hypothalamus because it contains neurons expressing Kiss1 and Tac3, two genes essential for reproduction and fertility. We chose the non-pathogenic adeno-associated virus (AAV) as a vector whose tropism could be modified to target the hypothalamus. The issues that must be overcome to utilize this vector as a delivery vehicle to induce sterility include modification of the wild-type AAV to target the hypothalamic region of the brain with a simultaneous reduction in targeting of peripheral tissues and non-hypothalamic brain regions, identification of RNAi targets that will effectively reduce the expression of Kiss1 and Tac3 without off-target effects, and determination if neutralizing antibodies to the AAV serotype of choice are present in cats. Successful resolution of these issues will pave the way for the development of a powerful tool to induce the permanent sterility in cats.
- Effect of testicular tissue lysate on developmental competence of porcine
oocytes matured and fertilized in vitro
- Authors: AK Singh; S Naskar, B Saikia, Y Vashi, S Gupta, S Banik, MK Tamuli, V Pande, DK Sarma, SK Dhara
Abstract: The objective of the present study was to investigate the effect of testicular tissue lysate (TTL) on developmental competence of germinal vesicle (GV) stage porcine oocytes. Two types of TTL were prepared through repeated freeze–thaw in liquid nitrogen, one from whole testicular tissue (wTTL) and other from either of four different sections of testes, namely just beneath the tunica albuginea (TA), from the transitional area between the seminiferous cord/tubules and the mediastinum testis (TR) and from the intermediate area (parenchymal tissue origin) and CE (cauda epididymis origin). The whole or section-wise TTL treatments were given for 44 hr during in vitro maturation (IVM). Oocyte maturation was done in either of the two media, namely defined (high-performance basic medium for porcine oocyte maturation, commercially available) and serum containing (TCM199). After maturation, oocytes were co-incubated with fresh spermatozoa for 6 hr and then transferred to embryo culture media. Treatment of GV stage oocytes with wTTL (1 mg/ml) increased the cleavage and morula percentage rate (69.23 ± 6.23 and 48.15 ± 6.77, respectively) than that of their control (58.33 ± 8.08 and 32.54 ± 5.53, respectively) in defined media, and in serum-containing media, cleavage and morula percentage rate were almost equal in both treatment (54.56 ± 7.79 and 34.70 ± 6.78, respectively) and control (59.52 ± 8.21 and 38.52 ± 6.54, respectively). However, effect of wTTL was not significant. In case of section-wise TTL supplements, TR section significantly (p
- Age and anti-Müllerian hormone levels predict the success of in vitro
maturation of cat oocytes
- Authors: F Snoeck; S Sarrazin, E Wydooghe, A Van Soom
Abstract: Up to date, in vitro maturation (IVM) rates of oocytes are highly variable between individual cats. This study was carried out to investigate the predictive value of age and anti-Müllerian hormone (AMH) concentration in relation to capacity for IVM of cat oocytes. Ovaries were collected from 33 cats, which were divided into three age groups: (i) 0–3 months (pre-pubertal); (ii) 3–12 months (peripubertal); and (iii) older than 12 months (pubertal). The cumulus–oocyte complexes (COCs) were matured and subsequently stained to check nuclear maturation status, and blood was taken for AMH analysis. Increasing age was significantly associated with decreasing AMH levels, and mean AMH levels differed significantly between all age categories: group 1: mean AMH 18.71 μg/L; group 2: mean AMH 9.27 μg/L; and group 3: mean AMH 4.13 μg/L. Moreover, the probability of maturation was more likely in groups 2 and 3 compared to group 1. Between categories 2 and 3, no significant difference in maturation probability was found (p = .31). Finally, the probability of oocyte maturation decreased significantly with increasing AMH levels. In age group 2, oocytes with a higher AMH level were less likely to mature. In age groups 1 and 3, no significant association between the AMH level and the proportion of maturated COC was found. We can conclude that if a higher probability of nuclear maturation is required, it is preferable to use cats with lower AMH levels and older than 3 months of age to improve cat IVM.
- Natural and artificial hyperimmune solutions: Impact on health in puppies
- Authors: H Mila; A Grellet, C Mariani, A Feugier, B Guard, J Suchodolski, J Steiner, S Chastant-Maillard
Abstract: Colostrum and milk are complex mammary secretions providing the puppy with many nutritional and immunological factors, which play a crucial role for its correct development and survival. In the case of colostrum and/or milk intake deficiency, puppies are at increased risk of infectious diseases. This work reviews the various nutritional hyperimmune supplementations proposed to provide a passive immune protection and to positively impact puppies’ health. Some strategies rely on canine immunoglobulins: canine colostrum banking and canine serum/plasma supplementation. Others involve heterologous sources of antibodies and other immune factors: bovine colostrum or hyperimmune egg powder. Among the different solutions evaluated from birth to weaning, canine plasma and hyperimmune egg powder showed promising beneficial effect on puppies’ health. Canine plasma seems to positively impact not only growth (increased growth during the neonatal period), but also digestive health (higher species richness of intestinal microbiota) and the general health (tendency of lower morbidity). Puppies supplemented with hyperimmune egg powder presented increased neonatal growth and decreased risk of canine parvovirus infection. Nevertheless, natural canine maternal colostrum and milk ingestion remains the optimal guarantee for puppies’ health and survival, as a source of immunity, energy and growth factors.
- Research on reproduction is essential for captive breeding of endangered
- Authors: K Jewgenow; BC Braun, M Dehnhard, J Zahmel, F Goeritz
Abstract: Assisted reproductive technology (ART) has great potential for conservation, but its successful application in captive breeding programmes of endangered species is often compromised by limited background on species' biology. Although carnivore species benefit from knowledge obtained in domesticated species (dogs, cats and ferrets), the focus of research is different. In pet animals, research in reproduction has mainly been focused on ovarian function and contraception, although substantial progress has also been made in the field of in vitro embryo production, transgenic embryos and cloning to aid relevant medical models. In endangered species, however, research should focus on characterizing reproductive traits (cyclicity and seasonality) to unravel species-specific endocrine principles of reproduction physiology. Based on this knowledge, it is crucial to enhance the ability to manipulate female reproductive cycles, especially those of embryo recipients. Furthermore, research conducted on molecular and cellular mechanisms of gamete and embryo development, as well as on cryopreservation protocols of gametes and embryos, is required for successful implementation of advanced ART to wild carnivores. This review will provide a summary on the state of the art with focus on ART contributing to conservation breeding of endangered carnivores.
- The nuclear and developmental competence of cumulus–oocyte complexes is
enhanced by three-dimensional coculture with conspecific denuded oocytes
during in vitro maturation in the domestic cat model
- Authors: MG Morselli; GC Luvoni, P Comizzoli
Abstract: The objective of the study was to assess the efficacy of coculture with conspecific cumulus-denuded oocytes (CDOs) during in vitro maturation in a three-dimensional system of barium alginate microcapsules on the in vitro embryo development of domestic cat cumulus–oocyte complexes (COCs). In Experiment I, COCs were cocultured with conspecific CDOs or cultured separately in a 3D system for 24 hr of in vitro maturation, before assessing the meiotic progression. In Experiment II, the in vitro fertilization of COCs and CDOs was carried out with chilled epididymal spermatozoa and the presumptive zygotes were cultured in vitro separately for 7 days in 3D microcapsules before assesment of embryonic development. The results showed that the viability was maintained and that meiosis was resumed in the 3D culture system. The presence of CDOs during in vitro maturation improved the meiotic competence of the COCs, since the proportions of telophase I/metaphase II were higher than that in the groups cultured separately. The enrichment of the maturation system by companion oocytes also enhanced the ability of COCs to develop into embryos, and increased the percentages of morula and blastoycst stages. The COCs cocultured with CDOs developed at higher rates than the COCs cultured separately and the CDOs themselves. The beneficial effects of coculture with conspecific CDOs were presumably due to the paracrine action of some secreted factors that enhanced many molecular patterns related to the complex of cumulus oophorous cells. Further investigations to understand how the 3D microenvironment can influence the features of oocytes and embryos are required.
- Expression of steroidogenic enzymes and steroid receptors in foetal gonads
of domestic cat—Sex similarities and differences
- Authors: BC Braun; K Jewgenow
Abstract: Foetal gonads already produce steroid hormones and by this influence the further development of external and internal genitalia as well as of the brain. Beside this, foetal gonads themselves can be influenced by foetal or maternal hormones. The time course of foetal gonadal development can differ between species. As knowledge on processes in domestic cats is very limited, the steroidogenic enzyme expressions as well as these of steroid receptors were analysed in foetal gonads of domestic cats. We investigated a period from beginning of the second half of pregnancy to the beginning of the third trimester; a phase, where also gonadal development proceeds. The mRNA expression of most of the steroidogenic enzymes was remarkably higher in male gonads compared to female ones on all analysed days. The enzyme mRNA expression in female gonads shows a tendency for an increase towards the beginning of the third trimester, except that of aromatase gene CYP19A1—it shows the opposite trend. CYP19A1 was detectable just in female gonads, indicating that only female foetal gonads are capable of producing oestrogens. Gene expressions of genomically and non-genomically acting steroid receptors for progesterone, androgen and oestrogen reception were observed in gonads of both genders. Slightly higher expressions of some receptors were detected in female compared to male gonads; only for the non-genomically oestrogen receptor GPER, we observed the opposite. The protein staining for progesterone receptor membrane component 1 (PGRMC1) exposed a potential function of it on steroid-producing cell and/or cells that suppose early oogenesis.
- Survival rate after vitrification of various stages of cat embryos and
blastocyst with and without artificially collapsed blastocoel cavity
- Authors: M Ochota; B Wojtasik, W Niżański
Abstract: Embryo vitrification is a modern technique for cryopreservation in assisted reproductive programs. From all the embryos, blastocysts are the most challenging during cryostorage due to their size, multicellular structure and the presence of blastocoelic fluid. The aim of this study was to evaluate the suitability for vitrification of various developmental stages of feline embryos and the influence of the artificial shrinkage (AS) of expanded blastocyst on post-vitrification survival rates. The AS procedure is the manual puncture of the trophectoderm allowing for the reduction of blastocoelic fluid prior to vitrification and thus preventing the ice crystal formation. The vitrified embryos were divided into groups of 2-cell, 4- to 8-cell, >8-cell, morulae, compacted and expanded blastocyst, based on morphological assessment and vitrified in groups of 1–3 embryos per Cryotop. The post-warming survival was similar regardless the embryo developmental stage prior vitrification; however, development to blastocysts was only noted in 4- to 8-cell and >8-cell vitrified embryos (13% and 27%, respectively). Following warming, the significantly more viable blastocysts were noted in vitrified compacted versus expanded blastocyst and in expanded blastocyst subjected to AS procedure versus expanded blastocyst without AS (total survival: 58.3% vs. 33.3% and 64.3% vs. 38.5%, respectively; re-expansion rate within 2 hr post-warming: 41.7 vs. 6.7% and 50% vs. 7.7%, respectively). One-fifth of vitrified expanded blastocyst showed morphological damage immediately after warming procedure, whereas no visible damage was noted in compacted blastocyst and artificially collapsed expanded ones. The obtained results suggest that the most suitable for vitrification are feline embryos containing four to 16 blastomeres and compacted blastocyst. In addition, the reduction of blastocoel cavity in expanding blastocyst by artificial collapse improved the blastocyst vitrification outcome.
- Safety and effectiveness of a single and repeat intramuscular injection of
a GnRH vaccine (GonaCon™) in adult female domestic cats
- Authors: LM Vansandt; MA Kutzler, AE Fischer, KN Morris, WF Swanson
Abstract: Sterilization is a key strategy to reduce the number of domestic cats entering and killed in shelters each year. However, surgical sterilization is expensive and labour-intensive and cannot fully address the 70 million free-roaming cats estimated to exist in the United States. GonaCon™ is a gonadotropin-releasing hormone vaccine originally developed for use as a wildlife immunocontraceptive. An earlier formulation was tested in domestic cats and found to be safe and effective for long-term contraception. However, the current Environmental Protection Agency (EPA)-registered formulation consists of a different antigen-carrier protein and increased antigen concentration and has never been tested in cats. A pilot study was undertaken to evaluate the short-term safety of a single GonaCon immunization, assess the consequences of vaccinated cats receiving an accidental second GonaCon injection and determine the humoral immune response to immunization. During Phase 1, cats in Group A (n = 3) received a single intramuscular injection of GonaCon and Group B (n = 3) received a single intramuscular injection of saline. During Phase 2, Group A received a second GonaCon injection and Group B received their initial GonaCon injection. All cats developed GnRH antibodies within 30 days of vaccine administration. The endpoint titre (1:1,024,000) was similar among all cats, and levels remained high throughout the duration of the study. Four cats developed a sterile, painless, self-limiting mass at the site of injection. The mean number of days to mass development was 110.3 (range, 18–249 days). In conclusion, this preliminary study suggests that the EPA-registered GonaCon formulation is safe for continued testing in domestic cats, an accidental revaccination should not increase the risk of a vaccine reaction and the EPA-registered formulation effectively elicits a strong humoral immune response.
- Decidualization of the canine uterus: From early until late gestational in
vivo morphological observations, and functional characterization of
immortalized canine uterine stromal cell lines
- Authors: FR Graubner; IM Reichler, NA Rahman, R Payan-Carreira, A Boos, MP Kowalewski
Abstract: The apparent lack of classical mechanisms for maternal recognition of pregnancy is one of the most intriguing features of canine reproduction. Consequently, similar levels of circulating luteal steroids are observed in pregnant and non-pregnant dogs. However, the early pre-implantation canine embryo locally modulates uterine responses to its presence, facilitating the successful onset of pregnancy. As a part of this interaction, the canine uterus undergoes a species-specific decidualization. Maternal stroma-derived decidual cells develop, the only cells of the canine placenta expressing progesterone receptor (PGR). There exists an acute need for an in vitro stable cell line model for canine decidualization. Therefore, herein our goal was to establish, immortalize and characterize such a cell line. We immortalized three monolayer dog uterine stromal (DUS) cell lines by stably transfecting them with SV40Tag oncogene. Cells retained their mesenchymal character for over 30 passages, as evidenced by VIMENTIN staining. Genomic incorporation of the SV40Tag protein was confirmed by immunofluorescence and Western blot analyses. Cells submitted to a classical in vitro decidualization protocol (N6,2′-O-dibutyryladenosine-3′,5′-cyclic monophosphate) revealed upregulated gene levels of selected major decidualization markers (e.g. PRLR, PGR, IGF1, PTGES). Additionally, the basic decidualization capability of PGE2 was demonstrated, revealing increased levels of, for example, PGR and PRLR gene expression, thereby implying its involvement in the progesterone-dependent decidualization in the canine uterus. In summary, our in vitro model with immortalized DUS cell line could serve as an ideal and unique model to study the underlying molecular and endocrine mechanisms of canine decidualization.
- Nasal immunization with inhibin DNA vaccine delivered by attenuated
Salmonella choleraesuis for improving ovarian responses and fertility in
- Authors: Q Liu; ZU Rehman, JJ Liu, L Han, XR Liu, LG Yang
Abstract: This study was conducted to determine the effect of immunization with inhibin DNA vaccine delivered by attenuated Salmonella choleraesuis on ovarian responses and fertility in cross-bred buffaloes. A total of 134 cross-bred buffaloes were divided into four groups: groups T1 (n = 34), T2 (n = 35) and T3 (n = 31) were nasal immunized twice a day with 10 ml of 1 × 1010 CFU/ml of the C501 (pVAX-asd-IS) vaccine for 5, 3 and 1 day, respectively. Group C (n = 34) was nasal immunized with 10 ml PBS for 5 days. All animals were immunized twice with an interval of 14 days and administered with 200 μg of a GnRH analogue on day 28, 0.5 mg PGF2α on day 35 and 200 μg of the same GnRH analogue on day 37. TAI was performed at 18 and 24 hr after the second GnRH treatment. Fourteen days after primary immunization, C501 (pVAX-asd-IS) elicited significant immune responses, and anti-inhibin IgG antibody titres in group T1 were significantly higher (p
- Uterine infection with bovine herpesvirus type 4 in dairy cows
- Authors: S Klamminger; I Prunner, MJ Giuliodori, M Drillich
Abstract: Diseases of the reproductive tract are a frequent problem in dairy herds. Herpesviruses are uterine pathogens also involved in other clinical diseases; for example, bovine herpesvirus type 4 BoHV-4 induces abortion, enteritis, metritis, pneumonia and vaginitis, but it can also be detected in healthy cows. The role of BoHV-4 in the development of clinical endometritis (CE) or subclinical endometritis (SE) has not clearly been described. Therefore, the objective of this study was to describe the prevalence of uterine BoHV-4 infection and its relationship with clinical, bacteriological and cytological findings in dairy cows 20–30 days after calving. The experiment was performed as a completely randomized block design, with farm (n = 10) as blocking criterion and with cow (n = 397) as the experimental unit. Logistic regression models were used to assess the effect of BoHV-4 infection on CE, SE and reproductive performance. Proportion of cows infected with BoHV-4 was 5.8% (n = 23/397). BoHV-4 was isolated in 11.0% (n = 12/109), 4.8% (n = 4/84) and 3.6% (n = 7/194) of cows diagnosed as CE, SE or healthy, respectively. A logistic model revealed that BoHV-4 infection showed a tendency to increase the risk for CE (AOR = 2.17; p = .10) but significantly reduced both, the odds for artificial insemination within 80 days post-partum (dpp) (AOR = 0.37; p = .035) and for pregnancy within 200 dpp (AOR = 0.13; p = .004). Furthermore, BoHV-4 infection increased the chance for intrauterine infection with Trueperella pyogenes (AOR = 5.55; p
- Estimation of endogenous levels of osteopontin, total antioxidant capacity
and malondialdehyde in seminal plasma: Application for fertility
assessment in buffalo (Bubalus bubalis) bulls
- Authors: P Kumar; M Saini, D Kumar, A Bharadwaj, PS Yadav
Abstract: This study was attempted to identify subfertile bulls by quantifying the endogenous levels of osteopontin (OPN), total antioxidant capacity (TAC) and malondialdehyde (MDA) in seminal plasma of buffalo bulls. On the basis of conception rate, buffalo bulls were classified into two groups: high-fertile (conception rate >50%) and subfertile bulls (conception rate
- The impact of cross-breeding Egyptian and Italian buffalo on reproductive
and productive performance under a subtropical environment
- Authors: MAF Nasr
Abstract: The aim of this study was to investigate the reproductive and productive performance of pure Egyptian (PE) buffaloes and their crosses with Italian buffaloes. In this study, 2969 dairy buffaloes were used (1599 PE; 615 F1 crosses, 50% PE and 50% Italian buffaloes; and 755 backcross [BC], 75% PE and 25% Italian buffaloes). When compared to PE, the BC and F1 had a significantly lower incidence of calving difficulty (odds ratio [OR] = 0.18, p
- Fertility of Angus cross beef heifers after GnRH treatment on day 23 and
timing of insemination in 14-day CIDR protocol
- Authors: RK Kasimanickam; JB Hall, WD Whittier
Abstract: This study compared artificial insemination pregnancy rate (AI-PR) between 14-day CIDR-GnRH-PGF2α-GnRH and CIDR-PGF2α-GnRH synchronization protocol with two fixed AI times (56 or 72 hr after PGF2α). On day 0, heifers (n = 1311) from nine locations assigned body condition score (BCS: 1, emaciated; 9, obese), reproductive tract score (RTS: 1, immature, acyclic; 5, mature, cyclic) and temperament score (0, calm; and 1, excited) and fitted with a controlled internal drug release (CIDR, 1.38 g of progesterone) insert for 14 days. Within herd, heifers were randomly assigned either to no-GnRH group (n = 635) or to GnRH group (n = 676), and heifers in GnRH group received 100 μg of GnRH (gonadorelin hydrochloride, IM) on day 23. All heifers received 25 mg of PGF2α (dinoprost, IM) on day 30 and oestrous detection aids at the same time. Heifers were observed for oestrus thrice daily until AI. Within GnRH groups, heifers were randomly assigned to either AI-56 or AI-72 groups. Heifers in AI-56 group (n = 667) were inseminated at 56 hr (day 32 PM), and heifers in AI-72 group (n = 644) were inseminated at 72 hr (day 33 AM) after PGF2α administration. All heifers were given 100 μg of GnRH concurrently at the time AI. Controlling for BCS (p
- miRNA and piRNA expression profiles of breeder cock testes detected by
- Authors: SR Wu; W Guo, YL Li, XC Ren, XY Lei, XY Li, JH Yao, XJ Yang
Abstract: miRNAs are small non-coding regulatory RNAs that play key roles in diverse biological processes. In this study, we used the Solexa sequencing technique to profile miRNAs in breeder cock testes to illustrate their functions. A total of 663 co-expressed miRNAs and 3,180 co-expressed piRNAs were detected in three libraries. Based on Mir-X™ miRNA qRT-PCR, three miRNAs representing low, medium and high expression levels according to the sequencing results were selected randomly to validate the miRNAs' expression profiles. Results suggested that the miRNA expression profiles data could represent actual miRNA expression levels. Moreover, target genes prediction of the co-expressed miRNAs and further Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed, which revealed that some candidate miRNAs were involved in the regulation of the spermatogenesis process, spermatozoa function and testicular metabolism. In conclusion, we provided a useful resource for further elucidation of the miRNAs' regulatory role in spermatogenesis, contributing to a preliminary database for functional and molecular mechanistic studies in testicular metabolism, spermatogenesis and other testes functions.
- Infertility associated with the absence of endometrial progesterone
receptors in a bitch
- Authors: JC Dockweiler; B Cossic, CG Donnelly, RO Gilbert, E Buckles, SH Cheong
Abstract: A three-year-old intact female Old English sheepdog was presented for evaluation of infertility. A uterine biopsy was performed during dioestrus, and the microscopic appearance was inconsistent with progesterone stimulation; the glands were sparse, simple and failed to show coiling, while the glandular epithelium was cuboidal instead of columnar. There was very little evidence of glandular activity. Due to the inappropriate appearance of the glands for the stage of the cycle, immunohistochemistry for progesterone receptors was performed. No progesterone receptor-positive immunoreactivity was identified in the endometrial luminal epithelium, glandular epithelium or stroma. Weak intranuclear immunoreactivity was identified within the smooth muscle cells of the myometrium. The absence of progesterone receptors within the endometrial glands is the most likely explanation for the abnormal appearance of the endometrium and for this bitch's infertility. To our knowledge, this is the first report of endometrial progesterone receptor absence in a bitch.
- Use of the gonadotropin-releasing hormone (GnRH) stimulation test to
monitor gonadal function in intact adult male cats
- Authors: S Romagnoli; A Baldan, C Righetti, C Fontaine, L Scenna, T Badon, C Stelletta, C Milani, M Cecchetto, A Mollo
Abstract: The gonadotropin-releasing hormone (GnRH) stimulation test is a common procedure used to investigate normality of the pituitary-gonadal axis in mammals. There is very little information on the technique, its efficacy and side effects in small animals and in particular no information for male cats. In dogs, such test is performed by intravenous (IV) administration. With cats, the number of times the animal needs to be restrained for blood sampling should be the least possible. The purpose of this study was to assess efficacy and side effects of the GnRH stimulation test in tomcats comparing the IV with the intramuscular (IM) route of administration. A GnRH stimulation test was performed in eight adult tomcats through IM or IV administration of 50 μg gonadorelin. The response of the pituitary-gonadal axis was assessed by measuring serum testosterone on blood samples collected prior to and 1 hr following treatment. When considering each single group of cats, the post-stimulation serum testosterone values were significantly higher than the pre-treatment ones (p
- Identification and changes in the seasonal concentrations of heat shock
proteins in roe deer (Capreolus capreolus) epididymides
- Authors: AM Majewska; W Kordan, M Koziorowska-Gilun, P Wysocki
Abstract: Heat shock proteins (HSPs) act as molecular chaperones with important regulatory functions. HSPs are considered to be essential factors in animal reproduction. In view of seasonal variations in the secretory activity of the reproductive tract of mature roe deer (Capreolus capreolus), the aims of this study were to identify HSPs in the epididymides and compare the expression of the identified proteins in three periods of the reproductive season. Two-dimensional polyacrylamide gel electrophoresis revealed the highest number of polypeptides in homogenates of epididymal tissues and in caput, corpus and cauda epididymal fluids throughout the reproductive season. Epididymal tissue homogenates and epididymal fluids were analysed by tandem mass spectrometry (MS/MS) to reveal 31 polypeptides with enzymatic activity, including polypeptides with antioxidant properties, structural and cell signalling functions. Moreover, among the identified polypeptides, five of them were similar to heat shock proteins: endoplasmin (Grp94); heat shock protein 90 kDa (HSP90); 78-kDa glucose-regulated protein (Grp78); chain A, the crystal structure of the human HSP70 ATPase domain and heat shock protein beta-1 isoform X. The concentrations of the analysed polypeptides, expressed in optical density units (ODU), differed significantly (p ≤ .05) across the examined periods of the reproductive season. The highest ODU values for almost all analysed proteins were observed during the rutting period. The presence of HSPs in the epididymal tissues and fluids of roe deer in different periods of the reproductive season could indicate that those proteins play an important role in sperm maturation in the epididymis.
- Expression pattern of HIF1alpha and vasohibins during follicle maturation
and corpus luteum function in the bovine ovary
- Authors: B Berisha; D Schams, D Rodler, F Sinowatz, MW Pfaffl
Abstract: The aim of this study was to characterize expression patterns of hypoxia-inducible factor-1alpha (HIF1A) and vasohibin family members (VASH1 and VASH2) during different stages of ovarian function in cow. Experiment 1: Antral follicle classification occurred by follicle size and estradiol-17beta (E2) concentration in the follicular fluid into 5 groups (180 E2 ng/ml). Experiment 2: Corpora lutea (CL) were assigned to the following stages: days 1–2, 3–4, 5–7, 8–12, 13–16 and >18 (after regression) of oestrous cycle and of pregnancy (months 1–2, 3–4, 6–7, >8). Experiment 3: Cows on days 8–12 were injected with a prostaglandin F2alpha (PGF) analogue and CL were collected before and 0.5, 2, 4, 12, 24, 48 and 64 hr after PGF injection. Expression of mRNA was measured by qPCR, steroid hormone concentration by EIA and localization by immunohistochemistry. HIF1A mRNA expression in our study increases significantly in follicles during final maturation. The highest HIF1A mRNA expression was detected during the early luteal phase, followed by a significant decrease afterwards. In contrast, the mRNA of vasohibins in small follicle was high, followed by a continuous and significant downregulation in preovulatory follicles. The obtained results show a remarkable inverse expression and localization pattern of HIF1A and vasohibins during different stages of ovarian function in cow. These results lead to the assumption that the examined factors are involved in the local mechanisms regulating angiogenesis and that the interactions between proangiogenic (HIF1A) and antiangiogenic (vasohibins) factors impact all stages of bovine ovary function.
- Differential expression and activity of matrix metalloproteinases 2 and 9
in canine early placenta
- Authors: M Diessler; M Ventureira, R Hernandez, C Sobarzo, L Casas, C Barbeito, E Cebral
Abstract: The zonary and endotheliochorial dog placenta is the most invasive placenta of carnivores. The importance of matrix metalloproteinases (MMP) in placenta invasiveness has been determined in several mammals including species with haemochorial, epitheliochorial and endotheliochorial placentation. Regarding the latter, the expression of MMP enzymes has been studied in the cat and the mature canine placenta. The aim of this study was to analyse the expression and activity of MMP-2 and MMP-9 in the early dog placenta. Placentae from 18 to 30 days of pregnancy were collected from four bitches. Two placentae from each bitch were analysed. Placental tissue from one uterine horn was fixed in formaldehyde for immunohistochemistry, while marginal haematoma, labyrinth, non-implantative and implantative endometrium from the contralateral horn were immediately frozen in dry ice for the analysis of MMP expression (Western blot [WB]) and activity (zymography). MMP-2 and MMP-9 were evidenced in the labyrinth, maternal glands and marginal haematoma; this finding was directly correlated with levels of MMP expression by WB, and with the activity of MMP-2, mainly in the haematoma (the area of major remodelling of tissues). Thus, although MMP-9 is well expressed in the early canine placenta, it is not active. Given the important role of MMPs for invasiveness, maternal–foetal angiogenesis and the establishment of a correct foetal nutrition, the results are consistent with the findings in other species in which the MMP-2 activation precedes the MMP-9 one in early placentation.
- Different associations of cryoprotectants for testicular tissue of
prepubertal cats submitted to vitrification
- Authors: DBC Lima; TFP Silva, GB Morais, A Aquino-Cortez, JSAM Evangelista, FAF Xavier Júnior, DA Viana, LDM Silva
Abstract: The cryopreservation of testicular tissue is presented as the only alternative for the preservation of genetic material from prepubertal animals. However, this biotechnology is still being tested. The objective of this study was to evaluate the effect of different associations of cryoprotectants and the potential of cell proliferation after vitrification of testicular tissue of prepubertal cats. Five testicular pairs from five prepubertal cats were used, and each pair was divided into four fragments. Of these, one fragment composed of the control group (CG) and the rest were distributed in experimental groups according to the associations of cryoprotectants to be tested (dimethyl sulphoxide (DMSO)/glycerol (GLY); ethylene glycol (EG)/GLY) or DMSO/EG) in a final cryoprotectant concentration of 5.6 m. The fragments were submitted to vitrification, and after one week, fragments were heated and processed for histomorphological evaluation and quantification of nucleolar organizer regions (NORs). DMSO/GLY did not differ from CG and was superior to the other vitrified groups, as to cell separation and degree of shrinkage of the basal membrane. Concerning cell differentiation, visibility of the nucleus and nuclear condensation, all the vitrified groups were inferior to CG; however, DMSO/EG was inferior to DMSO/GLY and EG/GLY, which did not differ among themselves. CG was superior to all groups in quantification of NORs. DMSO/EG was inferior to all others, and there was no difference between DMSO/GLY and EG/GLY. The association DMSO/GLY presented the best preservation of tissue integrity and potential of cell proliferation after vitrification of the testicular tissue of prepubertal cats.
- Responsiveness of intraovarian dog follicles in vitro to epidermal growth
factor and vascular endothelial growth factor depends on ovarian donor age
- Authors: C Thongkittidilok; DE Wildt, N Songsasen
Abstract: We investigated the influence of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) on in vitro follicle development within ovarian cortices recovered from pre-pubertal (≤6 months) versus peri-pubertal dogs (≥10 months). Ovarian cortices were cultured for 3 or 7 days in EGF (0 or 10 ng/ml) and VEGF (0, 0.1 or 1 ng/ml) and subjected to histological and apoptosis analyses. Fresh cortices from the same dogs served as “non-cultured controls” (NCC) and were evaluated similarly. The response of ovarian follicles to growth factors differed between pre-pubertal versus peri-pubertal tissues. For pre-pubertal dogs, percentage of structurally normal follicles in cortices cultured for 3 days in low VEGF (0.1 ng/ml) and EGF alone was comparable to that of the NCC. Follicle density declined in all cultured groups even after 3 days. Primary follicle diameter in all cortices cultured for 7 days, except in low VEGF, was smaller than that of the NCC, and percentage apoptotic follicles sharply increased in all treatment groups compared to the NCC. For peri-pubertal donors, percentages of structurally normal follicles decreased in all culture treatments at 3 and 7 days of incubation compared to the NCC. However, more normal follicles were found in cortices cultured in low VEGF and the two VEGF and EGF combinations than in the absence of growth factors or with EGF alone. Culture reduced the density of developing follicles, but follicle diameter was similar to that of the NCC. TUNEL analysis revealed that high-VEGF (1 ng/ml) treatment protected follicles against apoptosis, with the proportion of apoptotic follicles at Day 7 being comparable to that of the NCC. The findings demonstrate that the response of ovarian cortices to growth factor supplementation varied between pre-pubertal versus peri-pubertal donors.
- Evaluation of different fragment sizes and cryoprotectants for
cryopreservation of feline testicular tissues
- Authors: BI Macente; GH Toniollo, M Apparicio, CFM Mansano, HE Thomé, CL Canella, MEG Tozato, RR Gutierrez
Abstract: This study aimed to evaluate tissue damage of feline testicles sectioned in two different sizes (0.3 or 0.5 cm3) and submitted to different cryoprotectants (propanediol or glycerol). Testicles obtained from 12 domestic cats were sectioned in 0.3 and 0.5 cm3 sized pieces and immediately evaluated by TBARS and semi-quantitatively by histomorphology. The remaining fragments were placed in cryotubes with 1 ml Egg yolk Tris Equex STM extender containing 3% glycerol or 3% propanediol and cryopreserved by fast-freezing technique. Frozen-thawed fragments were also evaluated by TBARS and histomorphology. Statistical analysis was performed using one-way ANOVA with Student–Newmann–Keuls post hoc test, with p
- Reproductive performance and pre-weaning mortality: Preliminary analysis
of 27,221 purebred female dogs and 204,537 puppies in France
- Authors: S Chastant-Maillard; C Guillemot, A Feugier, C Mariani, A Grellet, H Mila
Abstract: The objective of this study was to describe efficiency of reproduction of purebred dogs in field breeding conditions, from mating to weaning in France. Data were collected between 2010 and 2014 in 5,667 French breeding kennels via a reproduction management software (Breeding Management System, Royal Canin, Aimargues, France). Effect of breed size (Mini: adult body weight 40 kg), age of dam and male on pregnancy rate, abortion rate and litter size were evaluated by multivariable models. Data on 45,913 heats (all with mating), from 27,221 bitches from 248 breeds, were analysed. At mating, mean age (±SD) was 3.1 ± 1.8 years for bitches and 3.3 ± 2.0 for males. Males originated from the same kennel as the females in 88.5% of the matings. Based on breeder's evaluation of the pregnancy status, pregnancy rate (number of pregnant females based on breeders declaration/number of heats) was 87.8% and abortion rate was 6.8%. Finally, 81.9% of the mated females gave birth to a litter. On 37,946 litters (204,537 puppies), mean litter size was 5.4 ± 2.8 puppies (range 1–24), which was influenced by breed size and dam age (p
- Molecular markers of putative spermatogonial stem cells in the domestic
- Authors: SJ Bedford-Guaus; S Kim, L Mulero, JM Vaquero, C Morera, R Adan-Milanès, A Veiga, Á Raya
Abstract: Spermatogonial stem cells (SSCs) are an important tool for fertility preservation and species conservation. The ability to expand SSCs by in vitro culture is a crucial premise for their use in assisted reproduction. Because SSCs represent a small proportion of the germ cells in the adult testis, culture success is aided by pre-enrichment through sorting techniques based on cell surface-specific markers. Given the importance of the domestic cat as a model for conservation of endangered wild felids, herein we sought to examine culture conditions as well as molecular markers for cat SSCs. Using a cell culture medium for mouse SSCs supplemented with glial cell-derived neurotrophic factor (GDNF), germ cells from prepuberal cat testes remained viable in culture for up to 43 days. Immunohistochemistry for promyelocytic leukaemia zinc finger (PLZF) protein on foetal, prepuberal and adult testis sections revealed a pattern of expression consistent with the labelling of undifferentiated spermatogonia. Fluorescence-activated cell sorting (FACS) with an antibody against epithelial cell adhesion molecule (EPCAM) was used to sort live cells. Then, the gene expression profile of EPCAM-sorted cells was investigated through RT-qPCR. Notably, EPCAM (+) cells expressed relatively high levels of CKIT (CD117), a surface protein typically expressed in differentiating germ cells but not SSCs. Conversely, EPCAM (-) cells expressed relatively high levels of POU domain class 5 transcription factor 1 (POU1F5 or OCT4), clearly a germ line stem cell marker. These results suggest that cat SSCs would probably be found within the population of EPCAM (–) cells. Future studies should identify additional surface markers that alone or in combination can be used to further enrich SSCs from cat germ cells.
- Epidemiological analysis of reproductive performances and kitten mortality
rates in 5,303 purebred queens of 45 different breeds and 28,065 kittens
- Authors: A Fournier; M Masson, F Corbière, H Mila, C Mariani, A Grellet, S Chastant-Maillard
Abstract: Reproduction management and performances are evaluated in the feline species only through a limited number of animals and studies. Our objective was to provide reference figures in purebred cats, from a large-scale sample. Data were collected from an online software dedicated to cattery management (Breeding Management System®, BMS, Royal Canin, Aimargues, France). Information was recorded on a voluntary basis by French breeders between 2011 and 2014. Data were anonymously transferred for analysis. A total of 9,063 oestrous periods (in contact with a male) from 5,303 queens (45 breeds) were recorded from 1,521 breeders. Most matings (70.1%) occurred during increasing day length periods. The mean age at mating (±SD) was 2.7 ± 1.6 years for queens and 2.9 ± 1.9 years for tomcats. Pregnancy rate (based on breeders declaration) was 85.2%. Among queens declared pregnant, 8.4% failed to maintain pregnancy. Globally, 78% of the mated females gave birth to 28,065 kittens within 7,075 L. Mean litter size was 4.0 ± 1.9 kittens among which 8.5% were stillborn. Neonatal and paediatric mortality rate was 8.2%. In total, 16.0% of kittens born died before weaning. The results of this study are based on the largest feline database ever analysed. The figures collected can thus be used as reference to define average reproductive performances in numerous breeds for cat breeders. Further analysis will identify factors influencing reproductive performances and early mortality in the feline species.
- Retained fertilizing capability in cryopreserved feline spermatozoa
- Authors: K Chatdarong
Abstract: Sperm cryopreservation offers a long-term preservation of male genetic materials for future assisted reproductive technologies. However, dramatic changes in temperature during freezing and thawing injure sperm cells. While motility is essential for AI and membrane integrity is crucial for in vitro fertilization (IVF), sperm DNA integrity is a common index of fertilizing capability required for AI, IVF and intracytoplasmic sperm injection (ICSI). In endangered felids died unexpectedly, attempts have been made to recover as many DNA intact spermatozoa as possible from epididymis and testis to increase the opportunity to produce offspring in future. Although sperm from caudal epididymis has shown retained fertilizing capability after freezing and thawing (27.3% conception rate after unilateral intrauterine insemination), sperm recovery from the corpus epididymis has been suggested as an alternative to increase the amount of preserved genetic materials. To improve epididymal sperm quality, pre-treatment with single-layer centrifugation resulted in selection of sperm cells with intact DNA while post-thaw treatment with extracellular ATP incubation promoted the blastocyst rate. Cold storage of domestic cat testis for 7 days at 4°C demonstrated
- Matrix-assisted laser desorption/ionization imaging mass spectrometry for
the spatial location of feline oviductal proteins
- Authors: M Apparicio; VG Santos, DFO Rocha, CR Ferreira, BI Macente, GM Magalhães, AE Alves, TF Motheo, LC Padilha-Nakaghi, EA Pires-Buttler, GC Luvoni, MN Eberlin, WRR Vicente
Abstract: With the purpose of identifying factors involved in early stages of embryo development in the domestic cat, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was used for the first time to describe the spatial localization of proteins in the oviducts of queens. Oviducts were obtained from two 2 and 4 years old cross-bred queens, divided into three segments, snap-frozen in liquid nitrogen and then stored at −80°C until use. Next, they were sectioned in a cryostat, fixed on ITO (indium tin oxide) conductive glass slides for MALDI-IMS and serial sections were collected on microscope slides for histology. As confirmed by histology, MALDI-IMS was able to show contrasting protein distributions in the oviductal infundibulum, ampulla and isthmus. Mass spectra were characterized by abundant ions of m/z 1,259, 4,939, 4,960 and 10,626, which have been tentatively attributed to keratin, thymosin β10, thymosin β4 and S100, respectively. Keratin and thymosins are involved in the biological response to tissue damage. S100 proteins are calcium-modulated proteins implicated in a variety of cellular activities, including cell differentiation and regulation of cell motility. These results suggest that protein composition differs between segments of the cat oviduct, which corresponds to morphological changes within these sections. Further functional studies could elucidate the effects of these proteins on feline reproductive physiology.
- Viability and growth of feline preantral follicles in vitro cultured with
insulin growth factor and epidermal growth factor supplemented medium
- Authors: AE Alves; LC Padilha-Nakaghi, EA Pires-Butler, M Apparicio, NAM Silva, TF Motheo, WRR Vicente, GC Luvoni
Abstract: In vitro culture of ovarian preantral follicles has emerged as a reproductive technology aimed at obtaining large amount of oocytes for in vitro embryo production. The addition of growth factors (GF) in the in vitro culture of preantral follicles of different species has provided superior results of follicular development, antrum formation and proliferation of granulosa cells. However, there are only few reports regarding the use of these factors on feline preantral follicle in vitro culture. Thus, the aim of this study was to investigate the effect of a combination of IGF-1 and EGF on in vitro viability and growth of preantral follicles and enclosed oocytes collected from domestic cats. A total of 64 follicles characterized by multilayer granulosa cells were isolated and individually cultured for 6 days (T6) in minimum essential medium supplemented with IGF-1+ EGF (100 ng/ml each) or without (control). A higher percentage of follicles were viable after culture with GF than without, and an increase in size when IGF-1+ EGF were added to the medium (170 ± 32.4 μm (T0) vs. 201 ± 22.3 μm (T6); p
- Canine pyometra: What is new?
- Authors: R Hagman
Abstract: Pyometra is a common disease in countries where elective spaying is not routinely performed. Hormonal and bacterial factors are fundamental in the pathogenesis of the disease, which manifests itself as a potentially life-threatening bacterial infection of the uterus. Surgical ovariohysterectomy is the safest and most effective treatment for pyometra, and it has recently been shown that laparoscopically assisted methods for surgical treatment are feasible to use in selected cases. New protocols for improved medical treatment alternatives have also been tested with promising results. To be able to predict outcome and presence of complications early would be valuable in clinical practice for optimizing therapy and increasing survival. Results of commonly investigated clinical and laboratory investigations have been shown to be useful as predictive markers, with leucopenia being associated with increased risk of peritonitis as well as prolonged post-operative hospitalization after surgical treatment. A cage-side rapid and cost-effective diagnostic test would be highly valuable in clinical practice, and detection of pyometra-specific upregulated genes in the uterus and the corresponding products is a potential start in identifying novel markers suitable for such as test. The focus of the present review is to highlight recent findings on pathogenesis, prediction of outcome, diagnosis and treatment. Additionally, central research questions and suggestions for future investigations about several aspects of canine pyometra will be addressed.
- Lipid composition of the canine sperm plasma membrane as markers of sperm
- Authors: CF Lucio; MM Brito, DSR Angrimani, KRA Belaz, D Morais, D Zampieri, JDA Losano, MEOA Assumpção, M Nichi, MN Eberlin, CI Vannucchi
Abstract: The fatty acid composition of the sperm membrane is an important factor involved in the overall sperm quality, including motility. However, in the canine species, the exact composition of the plasma membrane is still unknown. Therefore, the purpose of this study was to evaluate the plasma membrane lipid composition of motile sperm cells and to compare it with asthenospermic samples, as an attempt to determine possible involvements of membrane lipids in dog sperm cell motility. The sperm-rich fraction of ten mature dogs was collected, and samples were subjected to density gradient centrifugation by Percoll®, in order to separate motile and asthenospermic samples. Processed semen samples were evaluated for sperm motility, plasma and acrosome membrane integrity, mitochondrial activity and susceptibility to oxidative stress. Lipid plasma membrane composition was identified by mass spectrometry (MALDI-MS). The motile sperm samples presented the following phospholipids in a high frequency in the plasma membrane: phosphatidylcholine 38:4 (composed of stearic and arachidonic fatty acids), phosphatidylcholine 36:1 (stearic and oleic fatty acids), phosphatidylethanolamine 34:4 (myristic and arachidonic fatty acids), glycerophosphatidic acid 36:4 (palmitic and arachidonic fatty acids), phosphatidylcholine 40:4 plasmanyl and phosphatidylcholine 40:5 plasmenyl. Furthermore, no lipid markers were found in the asthenospermic samples. Results also indicate that differences on plasma membrane composition between motile and asthenospermic samples are crucial factors for determining sperm motility, sperm functionality and susceptibility to oxidative stress. In conclusion, plasma membrane lipid composition varies considerable between motile and asthenospermic samples. Therefore, lipid markers of sperm motility can be considered, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylcholine plasmanyl, phosphatidylcholine plasmenyl and phosphatidic acid.
- Urethral catheterization and sperm vitrification for simplified semen
banking in felids
- Authors: WF Swanson; HL Bateman, LM Vansandt
Abstract: Semen banking of domestic cats and wild felids represents a vital resource for their long-term conservation, but current methods require access to advanced training and specialized equipment. A newer method of semen collection, urethral catheterization of medetomidine-treated cats, allows recovery of high sperm numbers, but it is unclear if this approach permits maximal sperm recovery or is feasible using less expensive alpha-2 agonists. Similarly, a newer sperm preservation approach, vitrification, offers advantages of simplicity and minimal equipment needs, but its efficacy in combination with urethral catheterization has not been investigated. Our specific objectives were to (i) evaluate sequential semen recovery with urethral catheterization and electroejaculation in domestic cats, (ii) assess the effectiveness of a weak (xylazine) versus strong (dexmedetomidine) alpha-2 agonist for inducing sperm release, and (iii) compare post-thaw sperm motility, acrosome status and fertilizing capacity of catheter-recovered samples after vitrification or straw freezing. Results indicated that electroejaculation following repeated catheterization allowed recovery of additional spermatozoa (range, 11–32 × 106 sperm/male) and that xylazine was ineffective for inducing meaningful sperm release (range, 0–0.4 × 106 sperm/male). Post-thaw motility and acrosome status of vitrified catheter samples did not differ (p > .05) from that of straw frozen samples. Preliminary results indicated that in vitro fertilization success (9/30, 30%) of vitrified catheter sperm did not differ (p > .05) from that observed with straw frozen samples (17/30, 57%). In conclusion, urethral catheterization of dexmedetomidine-treated cats allows recovery of substantial sperm numbers but electroejaculation still may be warranted for maximal sperm recovery. Xylazine is not suitable as an inexpensive alternative to dexmedetomidine for catheterization. Vitrification of catheter samples results in comparable post-thaw parameters to straw freezing and may be adequate for use with oviductal insemination procedures.
- Uterine haemodynamic, vascularization and blood pressure changes along the
oestrous cycle in bitches
- Authors: IB Nogueira; LL Almeida, DSR Angrimani, MM Brito, RA Abreu, CI Vannucchi
Abstract: Characterization of the bitch's reproductive physiology is of utmost importance for the development of new reproductive techniques and the diagnosis of reproductive diseases. In this respect, uterine B-mode ultrasonography has been employed in several studies; however, the focus on haemodynamic changes along the oestrous cycle is yet to be described. Thus, the aim of this study was to characterize haemodynamic changes (uterine vascularization, systemic arterial blood pressure and heart rate) throughout the oestrous cycle in bitches. For this purpose, ten Golden Retriever bitches were evaluated during an entire oestrous cycle, twice during each stage of the cycle. Uterine artery blood flow, velocity wave forms, haemodynamic parameters and vascularization were analysed by Doppler ultrasonography. Furthermore, uterine diameter, arterial blood pressure and heart rate were measured. Uterine artery pulsatility index at early prooestrus was significantly lower in comparison with early oestrus, mid- and late anoestrus. Uterine artery resistance index was higher at early oestrus when compared to late oestrus and uterine diameter was significantly higher during late prooestrus. Furthermore, mean arterial blood pressure was lower and heart rate was higher during late prooestrus in comparison with the other oestrous cycle stages. In conclusion, haemodynamic changes in the uterine artery, uterine diameter, systemic blood pressure and heart rate occur during the canine oestrous cycle. Specifically, there is an increase in uterine artery perfusion, uterine diameter and mean arterial blood pressure during prooestrus, while uterine blood flow diminishes during oestrus and anoestrus.
- Canine reproductive ultrasound examination for predicting future sperm
- Authors: GCW England; L Bright, B Pritchard, IM Bowen, MB Souza, LDM Silva, R Moxon
Abstract: The reproductive potential of male animals is commonly evaluated using a breeding soundness examination incorporating B-mode ultrasound examination of the testes and recently Doppler ultrasound examination of the testicular arteries. These techniques may detect testicular normality or pathology, and while some measured parameters are associated with semen quality at the time of ultrasound examination, few studies have investigated the relationship with future semen quality. We hypothesized that B-mode and Doppler ultrasound measurements would correlate with future semen quality. Within two studies, we investigated the relationship between ultrasound measured testicular volume, testicular echogenicity, testicular homogeneity, subjective assessment of the testicular parenchyma, testicular artery resistance index, and pulsatility index with subsequent semen quality. Fifty-five normal fertile dogs of which 29 had stable semen quality and 26 had a subsequent decline in semen quality were examined during a 6-month period commencing 62 days after the ultrasound examination. Statistical analysis showed that no ultrasound parameters were predictive of future total sperm output or percentage live normal sperm. However, mean testicular echogenicity was positively related to future sperm motility (t = 2.202, p = .039). We conclude that quantitative ultrasound assessment of the appearance of the testicular parenchyma has potential for the evaluation of future semen quality in dogs.
- Deciphering the mechanisms involving cenexin, ninein and centriolin in
sperm maturation during epididymal transit in the domestic cat
- Authors: T Rowlison; MA Ottinger, P Comizzoli
Abstract: The sperm centrosome is an essential organelle with a key role in organizing the sperm aster for proper syngamy and formation of the first mitotic spindle. The sperm cell acquires the functional capability during epididymal transit by incorporation of key factors. The objective of the study was to identify these key maturation proteins, such as ninein and centriolin as well as cenexin—a scaffold protein that serves to bind ninein and centriolin. Epididymal samples were dissected from 17 adult cat testes (>1 year old) and spermatozoa were extracted from the different regions, including rete testis, caput, corpus, cauda and vas deferens. Tissue samples and sperm cells were fixed separately in 4% paraformaldehyde before immunostaining with anticenexin, ninein or centriolin antibodies. Results showed that the proportion of sperm cells with cenexin localized at the centrosome progressively increased along the tract with the lowest percentage of stained cells in the testis (mean = 45%) and highest in the cauda (mean = 81%). Although not significant, the intensity of cenexin immunofluorescence in positive cells increased twofold from the testis to vas deferens. There was no significant difference in the proportion of sperm labelled with centriolin or ninein (ranges of 21%–26% and 33%–48% between segments, respectively) or the intensity (±58% and ±63% change as compared to testis, respectively). Cenexin may serve as a scaffold protein for centriolin and ninein, as the vast majority of spermatozoa only displayed colocalization of these proteins when cenexin was also present (mean = 85% and 91% colocalization, respectively). In summary, these results could be applied to future efforts to create an in vitro culture system capable of rescuing the impaired centrosome of an infertile male, with particular potential for wild felid conservation.
- The influence of benign prostatic hyperplasia on sperm morphological
features and sperm DNA integrity in dogs
- Authors: RB Flores; DSR Angrimani, BR Rui, MM Brito, RA Abreu, CI Vannucchi
Abstract: Benign prostatic hyperplasia (BPH) has a high incidence in older intact dogs. Due to the increased prostatic oxidative stress and hormonal imbalance of BPH, sperm damage can arise, such as sperm morphological alterations and DNA fragmentation. This study aimed to compare the reproductive potential of healthy dogs and those affected by benign prostatic hyperplasia. Ten dogs were assigned to two experimental groups: dogs without BPH (control; n = 5) and dogs diagnosed with BPH (n = 5), based on clinical signs and ultrasonographic findings. Three semen collections were performed from each dog within one month and analysed using computer-assisted sperm analysis (CASA) and functional tests. Control group showed higher percentage of sperm DNA integrity (95 ± 1.8%) compared to the BPH group (79.2 ± 6.4%). On the other hand, the percentage of minor sperm defects, amplitude of lateral sperm head displacement of the spermatozoa and medium sperm mitochondrial activity were higher in the BPH group. In conclusion, BPH decreases sperm DNA integrity, increases mitochondrial activity, as well as modifies sperm movement pattern. Therefore, a careful sperm analysis of aged dogs with BPH is required before a reproductive programme can be established for such patients.
- Kisspeptin-10 and the G protein-coupled receptor 54 are differentially
expressed in the canine pregnant uterus and trophoblast cells
- Authors: S Schäfer-Somi; SS Ay, D Kaya, M Sözmen, HB Beceriklisoy, AR Ağaoğlu, M Fındık, T Haeften, S Aslan
Abstract: Uterine tissue was collected from bitches after ovariohysterectomy at different times after ovulation. Samples were assigned to four groups: metestrous non-pregnant, day 10–12, n = 4; pre-implantation, day 10–12, n = 9; post-implantation, day 18–25, n = 13; mid-gestation, day 30–40, n = 7. RT-qPCR detection was performed for kiss1 and the G protein-coupled receptor 54 (GPR54, specific receptor for kisspeptin). In addition, immunohistochemistry was performed for detection of kisspeptin-10 (KP-10), GPR54, as well as pan-cytokeratin and vimentin. The latter two were included to differentiate the different placental cell types. The percentage of positive stained cells was evaluated, and an immunoreactivity score (IRS) was obtained by multiplying the labelling intensity score (0–3) with the percentage of immunolabelled cells (range: 0–300). In non-pregnant and pre-implantation tissues, gene expression was highly variable for kiss1 and GPR54. Expression of GPR54 was higher before embryo adhesion than during post-implantation and mid-gestation (p
- Characterization of teratogenic potential and gene expression in canine
and feline amniotic membrane-derived stem cells
- Authors: MT Cardoso; AO Pinheiro, AS Vidane, JB Casals, VC Oliveira, NJN Gonçalves, DS Martins, CE Ambrósio
Abstract: The biosafety of innovative procedures that utilize stem cells in regenerative medicine has been addressed in several studies. Previous work has showed no tumour formation following the use of feline and human amniotic membrane-derived stem cells (AMSCs). In contrast, tumour formation was observed when canine AMSCs were utilized. These findings suggested that feline and human, but not canine, AMSCs are suitable for cell transplantation trials. This study aimed to further evaluate the feasibility of utilizing canine AMSCs for transplantation purposes as well as for felines. We tested teratoma formation following cell injection into BALB/c nude mice and then assessed expression of haematopoietic, mesenchymal, tumorigenic, pluripotency and cellular regulation markers using flow cytometry and qPCR. The use of canine AMSCs did not result in macroscopic tumour formation as determined 60 days after transplantation. The immunophenotypic characterization by flow cytometry revealed expression of mesenchymal markers (CD73 and CD90) and expression of the pluripotent marker OCT4 and SOX2. Quantitative PCR analysis revealed that there were no differences in the patterns of gene expression (CD34, CD73, OCT4, CD30 and P53) between canine and feline AMSCs, with the exception of the expression of SOX2 and CD90.
- Lysophosphatidic acid expression in theca cells depends on the type of
bovine ovarian follicle
- Authors: E Sinderewicz; K Grycmacher, D Boruszewska, I Kowalczyk-Zięba, I Woclawek-Potocka
Pages: 28 - 34
Abstract: Lysophosphatidic acid (LPA) exerts various actions on the mammalian reproductive system. In cows, LPA stimulates the synthesis and secretion of luteotropic factors in the ovary, which affects the growth and development of ovarian follicles. The role of LPA in granulosa cells, oocyte and oocyte-cumulus complex (COC) has previously been investigated; but its role in the theca layer, which is an important structural and functional component of the ovarian follicle, is still unclear. The goal of this study was to investigate the expression of LPA in theca cells originating from different bovine ovarian follicle types. Theca cells were separated from healthy, transitional and atretic ovarian follicles, based on intrafollicular estradiol: progesterone ratios. LPA concentration in the follicular fluid (FF) in different follicle types was measured, and expression of the enzymes responsible for LPA synthesis (autotaxin [AX], phospholipase A2 [PLA2]) and receptors for LPA (LPAR1-4) were determined. The obtained results confirmed the follicle-type dependent presence of LPA in the FF of the bovine ovarian follicles. The highest concentration of LPA was detected in follicles classified as healthy and dominant. LPAR1-4, PLA2 and AX expression in theca cells in all of the types of follicles examined were detected at mRNA and protein level. These results suggest that theca cells can be a source of LPA synthesis other than granulosa cells and COCs, as well as the target for its action in the bovine ovarian follicle, with PLA2 and LPAR4 playing major roles in LPA synthesis and action.
- Effect of climate region and stocking density on ostrich (Struthio
camelus) productive performances
- Authors: M Bouyeh; A Seidavi, H Mohammadi, A Sahoo, V Laudadio, V Tufarelli
Pages: 44 - 48
Abstract: The effects of three climates (hot and dry, mild and humid and Alpine) and three flock densities (300 m2) on ostrich reproductive and productive traits were studied. Data were compared with the benchmark target sets by the World Ostrich Association (Ostrich benchmark Performance Targets. Version 2, May, 2008) for reproductive qualifications of ostrich. No significant difference was observed on egg production, weight, fertility, hatchability and day-old chicks weight among the three climate conditions; however, the Alpine climate had a lower performance trend. Mild and humid climates had a significant effect of age at sexual maturity for both males and females as well as on the duration of egg production season. Stocking density did not show significant difference on egg production, hatchability, age of male and female at sexual maturity and on duration of egg production season, while an area >300 m2 showed a reduction in egg weight and day-old chick weight. Further, an area
- Semen supplementation with palmitoleic acid promotes kinematics,
microscopic and antioxidative parameters of ram spermatozoa during liquid
- Authors: M Eslami; H Ghasemiyan, E Zadeh Hashem
Pages: 49 - 59
Abstract: The purpose of the present experiment was to investigate the protective effects of palmitoleate on the quality of ram semen during low temperature liquid storage. Ejaculates were collected using the artificial vagina from four Qezel rams twice a week. Ejaculates were pooled, diluted with Tris–egg yolk extender without palmitoleate (control) or supplemented with 0.125 (P 0.125), 0.25 (P 0.25), 0.5 (P 0.5) and 1 (P 1) mM palmitoleate at a final concentration of 500 × 106 spermatozoa/ml. Total motility and forward progressive motility (FPM) as well as other spermatozoa kinematics were evaluated by computer-assisted sperm analysis. Moreover, viability and membrane functionality were determined in the spermatozoa. Additionally, amounts of malondialdehyde (MDA), total antioxidant activity (AOA), nitric oxide (NO) and superoxide dismutase (SOD) activities were evaluated in the medium and spermatozoa at 0, 24, 48 and 72 hr of storage. The palmitoleate supplementation resulted in a significant (p
- Organogenesis and foetal haemodynamics during the normal gestation of
healthy black-rumped agoutis (Dasyprocta prymnolopha, Wagler, 1831) bred
- Authors: FCA Sousa; GT Pessoa, LS Moura, JR Araújo, RPS Rodrigues, MAPS Barbosa, AN Diniz, AB Souza, EG Silva, LU Lucena, MP Sanches, OF Silva-Filho, PC Guerra, JM Sousa, WC Neves, FR Alves
Pages: 60 - 66
Abstract: The objective of this study was to define the patterns of organogenesis and foetal haemodynamics during the normal gestation of healthy agoutis (Dasyprocta prymnolopha) kept in captivity. Thirty pregnant agoutis that ranged in size from small to medium and weighed between 2.5 and 3 kg underwent B-mode and Doppler ultrasonography for the biometric evaluation of the foetal organs. The foetal aortic blood flow proved to be predominantly systolic, and the measured flow velocity was 78.89 ± 2.95 cm/s, with a maximum pressure gradient of 2.12 ± 0.27 mmHg. The liver was characterized by its large volume, occupying the entire cranial aspect of the abdominal cavity, and it was associated cranially with the diaphragm and caudally with the stomach. The flow velocity in the portal vein was estimated to equal 12.17 ± 2.37 cm/s, with a resistivity index of 0.82 ± 0.05. The gallbladder was centrally located and protruded cranially towards the diaphragm. The spleen was visualized as an elongated structure with tapered cranial and caudal extremities, and the foetal kidneys were visualized bilaterally in the retroperitoneal region, with the right kidney positioned slightly more cranially than the left. The morphological characterization and hemodynamic analysis of the foetal organs of black-rumped agoutis via B-mode and Doppler ultrasonography allow determination of the vascular network and of reference values for the blood flow required for perfusing the anatomical elements essential for maintaining the viability of foetuses at different gestational ages.
- Detecting reproductive system abnormalities of broiler breeder roosters at
- Authors: MA Lagares; R Ecco, NRS Martins, LJC Lara, JSR Rocha, DAR Vilela, VM Barbosa, PF Mantovani, JFV Braga, IS Preis, VA Gheller, PC Cardeal, NC Baião
Pages: 67 - 75
Abstract: The objective of this study was to detect the reasons of rooster's fertility decrease at 50 weeks of age. Therefore, the reproductive system of broiler breeder roosters was laparoscopic, macroscopic and histopathology evaluated, and a comparison of the anatomical aspect with the sperm analysis and birds’ age was realized. Cobb roosters (n = 59) were distributed into two groups (30 and 50 weeks). Evaluations were performed with laparoscopy, macroscopy and histopathology, and seminal quality, blood serum testosterone concentration and weight were also determined. The old roosters presented smaller testicle size, higher intensity epididymal lithiasis and lower testicle sperm production, compared to the young roosters. The use of the endoscope could easily distinguish a normal-sized testicle than an atrophic one. Four old roosters with severe testicular atrophy did not show spermatogenesis, although three still had sperm in the ejaculate. This would falsely indicate a wrong diagnosis of normal fertility before the testicular atrophy took place. In conclusion, in addition to the weight increase with age, the testicular atrophy and impairment of sperm production seemed to be the main reason to the decrease in the rooster's fertility at 50 weeks of age. Therefore, the use of the laparoscopy as a way to detect the roosters with testicular atrophy before 50 weeks of age and their removal from them flock could be useful as a diagnostic tool to prevent the birds’ fertility loss.
- Fixed-time post-cervical artificial insemination in weaned sows following
buserelin use combined with/without eCG
- Authors: E Baroncello; ML Bernardi, AD Kummer, I Wentz, FP Bortolozzo
Pages: 76 - 82
Abstract: Fixed-time post-cervical artificial insemination (FTAI) drastically reduces labour requirements and increases the use of boars with higher genetic merit. This study evaluated the efficiency of eCG administration combined with/without the GnRH agonist buserelin for the induction and synchronization of ovulation in weaned sows submitted to FTAI. The sows were allocated into three groups. In the control group, the first artificial insemination was performed at the onset of oestrus and repeated every 24 hr. In the eCG+GnRH group, sows received 600 IU eCG at weaning and buserelin (10 μg) after 86–89 hr of eCG, and in the GnRH group, sows received only buserelin after 86–89 hr of weaning. The hormone-treated sows received a single FTAI after 30–33 hr of buserelin application. All the sows were inseminated with homospermic doses (1.5 × 109 sperm cells/50 ml). The interval between weaning and ovulation was shorter (p
- A three-dimensional alginate system for in vitro culture of
cumulus-denuded feline oocytes
- Authors: MG Morselli; S Canziani, D Vigo, GC Luvoni
Pages: 83 - 88
Abstract: In the case of high valuable individuals with very precious genetic material, widening the genetic pool including gametes with poor morphological characteristics, as cumulus-denuded oocytes (CDOs), could be an option. To improve the in vitro culture of low-competence feline CDOs, an enriched three-dimensional (3D) system in association with competent cumulus–oocyte complexes (COCs) was developed. For this purpose, domestic cat CDOs were cultured with or without companion COCs in the 3D barium alginate microcapsules. The overall viability and the meiotic progression of feline CDOs cocultured with COCs or cultured separately in 3D or in 2D (traditional microdrops) system were compared. The 3D system was able to support viability and meiotic resumption of the feline oocytes, as well as the 2D microdrops. In 3D microcapsules, the presence of COCs resulted in a higher viability of CDOs (91.1%, p .05). It is notable that the presence of CDOs seemed to enhance the meiotic progression of the associated COCs. In conclusion, the 3D barium alginate microcapsules are a suitable system for feline oocytes in vitro culture, but more specific enriched conditions should be developed to improve the CDOs full competence in vitro.
- The effect of select seminal plasma proteins on endometrial mRNA cytokine
- Authors: CE Fedorka; KE Scoggin, EM Woodward, EL Squires, BA Ball, MHT Troedsson
Pages: 89 - 96
Abstract: In the horse, breeding induces a transient endometrial inflammation. A subset of mares are unable to resolve this inflammation, and they are considered susceptible to persistent mating-induced endometritis PMIE Select seminal plasma proteins cysteine-rich secretory protein-3 (CRISP-3) and lactoferrin have been shown to affect the innate immune response to sperm in vitro. The objective of this study was to determine whether the addition of CRISP-3 and lactoferrin at the time of insemination had an effect on the mRNA expression of endometrial cytokines in susceptible mares after breeding. Six mares classified as susceptible to PMIE were inseminated during four consecutive oestrous cycles with treatments in randomized order of: 1 mg/ml CRISP-3, 150 μg/ml lactoferrin, seminal plasma (positive control) or lactated Ringer's solution (LRS; negative control) to a total volume of 10 ml combined with 1 × 109 spermatozoa pooled from two stallions. Six hours after treatment, an endometrial biopsy was obtained for qPCR analysis of selected genes associated with inflammation (pro-inflammatory cytokines interleukin (IL)-1β, IL-8, tumour necrosis factor (TNF)-α, interferon (INF)-γ, anti-inflammatory cytokines IL-1RN and IL-10, and inflammatory-modulating cytokine IL-6). Seminal plasma treatment increased the mRNA expression of IL-1β (p = .019) and IL-8 (p = .0068), while suppressing the mRNA expression of TNF (p = .0013). Lactoferrin also suppressed the mRNA expression of TNF (p = .0013). In conclusion, exogenous lactoferrin may be considered as one modulator of the complex series of events resulting in the poorly regulated pro-inflammatory response seen in susceptible mares.
- Differential expression of ISG 15 mRNA in peripheral blood mononuclear
cells of nulliparous and multiparous pregnant versus non-pregnant Bos
- Authors: NP Soumya; DN Das, S Jeyakumar, S Mondal, A Mor, UT Mundhe
Pages: 97 - 106
Abstract: Embryonic mortality is found to be the main source of reproductive wastage in domestic ruminants. Many genes are involved in the growth and development of the embryo, and the interferon-stimulated gene 15 (ISG 15) is one of the major gene stimulated by interferon tau, the maternal recognition of pregnancy signal in ruminants. In this study, both genomic and cDNA sequences of ISG 15 from Bos indicus (Deoni breed) were amplified and characterized. The genomic sequence of Deoni ISG 15 exhibited 99% identity with Bos taurus and 97% identity with that of Bos mutus and Bubalus bubalis. Moreover qRT-PCR analysis revealed constitutive expression of the ISG 15 mRNA in peripheral blood mononuclear cells of Deoni heifers and multiparous cows during early pregnancy. Fourteen Deoni heifers and fifteen multiparous Deoni cows were synchronized for timed AI by CIDR-Ovsynch protocol, and six animals were kept as cyclic control in each group. Blood samples were collected on days 7, 14, 16, 18, 21, 30 and 45 from the day of AI. Pregnancy was confirmed by plasma progesterone level through ELISA. A significantly higher expression of ISG 15 mRNA was found on day 16 (p
- Motility and oxidative–antioxidant capacity of Huso huso semen,
stored at −80°C
- Authors: MS Aramli; RM Nazari, S Aramli, HA Nouri
Pages: 170 - 173
Abstract: In this study, motility and oxidative–antioxidant capacity (thiobarbituric acid-reactive substance level [TBARS], superoxide dismutase [SOD], catalase [CAT] and glutathione reductase [GR]) of beluga sturgeon (Huso huso) sperm, stored for 6 days at −80°C, were evaluated. After 2 days of storage, sperm motility was significantly decreased (no motile sperm were observed after 6 days of storage; p