Secretome derived from different cell lines in bovine embryo production in vitro.

The present study investigated the effects of conditioned medium (CM), composed of microvesicles (MVs) and soluble factors present in the supernatant (SN), of bovine endometrial and amniotic cells on embryo quality and rate of blastocyst production. Presumptive zygotes were randomly assigned on Days 1, 3 and 5 after fertilisation to synthetic oviducal fluid with amino acids (SOFaa; control) or to SOFaa supplemented with either 20% endometrial or amniotic CM, 20% SN or 100×106MVsmL-1. Embryos were evaluated on Day 7. For groups supplemented with MVs derived from either endometrial or amniotic cells on Day 1 of culture, blastocysts had developed, but at a lower rate than in the control group. Blastocysts had developed in all groups in which endometrial or amniotic cell-derived CM or MVs were added on Day 3 of culture, but the rate of blastocyst development was significantly lower in both CM groups than in the MVs groups. The addition of all secretome fractions (CM, MVs and SN) derived from either bovine endometrial or amniotic cells on Day 5 of culture resulted in blastocyst production, but only amniotic MVs resulted in a blastocyst production rate comparable to that in the control group. Supplementation of SOFaa on Day 5 resulted in a qualitatively higher number of inner cell mass cells compared with the control group only for the amniotic CM and MVs groups. At day 7, these data were confirmed by RT-qPCR evaluation of genes (Bcl-2-associated X protein (BAX) and glutathione peroxidase 1 (GPX1) involved in apoptosis and protection against reactive oxygen species. In conclusion, of the different secretome fractions tested, only amniotic MVs added to SOFaa resulted in better outcomes than in the control group.

Intramammary administration of platelet concentrate as an unconventional therapy in bovine mastitis: first clinical application.

Bovine udder infections induce a variety of changes in gene expression of different growth factors that may suggest their possible role in glandular tissue protection or repair processes. Growth factors and also chemokines and cytokines may act synergistically to increase the infiltration of neutrophils and macrophages to promote angiogenesis, fibroplasia, matrix deposition, and, ultimately, re-epithelialization. Considering the vast applications, typically in human medicine, of platelet concentrate (PC) and its ease of preparation, the aim of our study was to evaluate an alternative therapy to stimulate the regeneration of glandular tissue, administering a concentration in excess of the growth factors contained in the PC. In each one of the 3 farms examined in the trial, PC was prepared from donor cows in good health, free from infections, and with no records of medications administered during the previous 2 mo. The platelet produced in one farm was used only for treating the cows of the same farm in a heterologous way. A total of 229 mastitic quarters were divided in 3 groups: antibiotic group (treated with intramammary antibiotic), antibiotic and PC group (treated intramammarily with antibiotics in association with PC), and PC group (treated with intramammary PC alone). The diagnosis of mastitis was based on somatic cell count and bacteriological evaluation of the milk from the affected quarter. Platelet concentrate, alone or in association with antibiotic, was used for 3 consecutive days as an unconventional therapy in bovine acute and chronic mastitis. Our data show that the associated action of antibiotic and PC performed significantly better than the antibiotic alone, either for the recovery of the affected mammary quarters or for somatic cell count reduction. In the same way, the association antibiotic plus PC showed significantly fewer relapses compared with the antibiotic alone, either for acute or chronic mastitis. The treatment with only PC did not show statistically significant differences compared with both antibiotic alone or associated treatment for acute mastitis, and it was better than the use of only antibiotic for chronic mastitis. Our results show that PC alone may be useful for a quick resolution of the inflammatory response, playing a role in limiting the tissue damage to the mammary gland parenchyma and reducing the recurrence rates.

A novel monoclonal antibody-based enzyme-linked immunosorbent assay to determine luteinizing hormone in bovine plasma.

The development of a novel enzyme-linked immunosorbent assay (ELISA) for determining luteinizing hormone (LH) in bovine plasma is described. Anti-bovine LH (bLH) monoclonal antibodies (mAbs) were produced and characterized. One mAb recognizing the bLH ß subunit was used for immunoaffinity purification of substantial amounts of biologically active bLH from pituitary glands. The purified bLH in combination with 2 anti-bLH ß subunit mAbs was used to develop a sandwich ELISA, which satisfied all the criteria required to investigate LH secretory patterns in the bovine species. The ELISA standard curve was linear over the range 0.05 to 2.5 ng/mL, and the assay proved suitable for measuring bLH in plasma without any prior treatment of samples. Cross-reactivity and recovery tests confirmed the specificity of the method. The intra- and inter-assay coefficients of variation ranged between 3.41% and 9.40%, and 9.29% and 15.84%, respectively. The analytical specificity of the method was validated in vivo by provocative tests for LH in heifers, using the LH releasing peptide gonadotropin-releasing hormone. In conclusion, the adoption of mAbs for this ELISA for coating the wells and labeling, combined with the easy one-step production of reference bLH, ensures long-term continuity in large-scale measurements of LH in the bovine species.

Acute follicular response to FSH in heifers downregulated long term with a GnRH agonist and with suppressed ovarian follicular growth.

The objectives of this study were to (1) ascertain ovarian follicular status in heifers where the pituitary gonadotrope cells producing LH and FSH were downregulated by long-term treatment with a GnRH agonist and (2) characterize the acute superstimulation of follicular growth in response to FSH in heifers where the pituitary was downregulated with a GnRH agonist. At the start of the study, heifers (3 year old) were implanted with GnRH agonist (n = 20) or received no treatment (control, n = 5). After 6 months, follicular growth in heifers implanted with GnRH agonist was restricted to early antral follicles (2-4 mm). At this time, these heifers were allocated to four groups and follicular development was superstimulated with FSH as follows: group 1 (n = 5) and group 2 (n = 5), a conventional FSH protocol with injections over 4 days (Days 0-3) with Group 2 receiving two times the normal dose of FSH; group 3 (n = 5), a single injection of FSH in saline on Day 0; group 4 (n = 5), a single injection of FSH in slow-release polyvinylpyrrolidone on Day 0. Follicular growth in the control heifers (n = 5) was superstimulated using the conventional 4-day FSH protocol. On Day 5, heifers in groups 1 and 2 had similar (P > 0.05) numbers of follicles in the size categories 6 to 7 mm (12.8 ± 3.0 follicles) and 8 to 9 mm (6.5 ± 1.0) as control heifers (6-7 mm, 14.6 ± 2.2; 8-9 mm, 6.2 ± 2.2) and six of the former heifers had follicles 10 mm (3.2 ± 1.4). Also on Day 5, follicular growth for heifers in group 3 was restricted to 4 to 5 mm (11.6 ± 3.0) and 6 to 7 mm (7.6 ± 3.7), whereas four out of five heifers in group 4 had follicles of 6 to 7 mm (8.2 ± 2.3) and three heifers had follicles of 8 to 9 mm (4.6 ± 2.2) and 10 mm (2.2 ± 0.9). Injection of exogenous LH on Day 5 induced ovulation in 9 out of 10 heifers in groups 1 and 2, no heifers in group 3, and 2 of 5 heifers in group 4. Plasma concentrations of progesterone 6 days after ovulation were the same (P > 0.05) for heifers in groups 1 and 2 (8.9 ± 0.9 ng/mL) and control heifers (10.0 ± 0.9 ng/mL). This study has shown that heifers treated chronically with a GnRH agonist have suppressed ovarian follicular growth but are able to respond to acute superstimulation with FSH. Furthermore, follicles in heifers treated with a GnRH agonist undergo morphologically normal growth in response to FSH and can ovulate and develop into a CL. The long-term GnRH agonist-treated heifer provides a practical model for repeated ovarian follicular superstimulation, multiple ovulation, and embryo transfer, without the need to control stage of the estrous cycle.

Characteristics of equine mesenchymal stem cells derived from amnion and bone marrow: in vitro proliferative and multilineage potential assessment.

REASONS FOR PERFORMING STUDY: This is the first study comparing stemness features of equine mesenchymal progenitor cells derived from amniotic membrane and bone marrow. OBJECTIVES: To investigate an alternative and noninvasive stromal cell source for equine tissue engineering. STUDY DESIGN: In vitro experimental study of the characteristics of equine mesenchymal progenitor cells derived from amnion and bone marrow. METHODS: Cells isolated from amniotic membrane and bone marrow were analysed for proliferation (growth curve, doubling time, colony forming unit). Immunocytochemical detection of pluripotency markers and gene expression of stromal cell markers were also performed and these cells were studied for multilineage plasticity. RESULTS: Amniotic stromal cells (AMSCs) and bone marrow mesenchymal cells (BM-MSCs) both exhibited mature stromal cell-specific gene expression and immunocytochemical properties, but showed substantial differences in their proliferative and differentiation potential. The mean doubling time for AMSCs was significantly lower (P<0.05) than that observed for BM-MSCs (1.17 ± 0.15 vs. 3.27 ± 0.19 days, respectively). Compared to AMSCs, BM-MSCs also demonstrated a significantly (P<0.05) lower clonogenic capability (one fibroblast-like colony forming unit from a mean of 590.15 cells seeded for BM-MSCs vs. 242.73 cells seeded for AMSCs). BM-MSCs did not differentiate into glial cells, and the osteogenic differentiation process was longer than for AMSCs. CONCLUSIONS AND POTENTIAL RELEVANCE: The amniotic membrane could be a valuable source of MSCs to be used both for allogenic and/or autologous therapies. The noninvasive nature and low cost of collection, the rapid proliferation along with a greater differentiation potential and the 'off the shelf' preparation potential could make AMCs useful for cell therapy.

Mesenchymal stem cells from amnion and amniotic fluid in the bovine.

Amnion and amniotic fluid (AF) are noncontroversial and inexhaustible sources of mesenchymal stem cells (MSCs) that can be harvested noninvasively at low cost. As in humans, also in veterinary field, presumptive stem cells derived from these tissues reveal as promising candidates for disease treatment, specifically for their plasticity, their reduced immunogenicity, and high anti-inflammatory potential. The aim of this work is to obtain and characterize, for the first time in bovine species, presumptive MSCs from the epithelial portion of the amnion (AECs) and from the AF (AF-MSCs) to be used for clinical applications. AECs display a polygonal morphology, whereas AF-MSCs exhibit a fibroblastic-like morphology only starting from the second passage, being heterogeneous during the primary culture. For both lines, the proliferative ability has been found constant over the ten passages studied and AECs show a statistically lower (P<0.05) doubling time with respect to AF-MSCs. AECs express MSC-specific markers (ITGB1 (CD29), CD44, ALCAM (CD166), ENG (CD105), and NT5E (CD73)) from P1 to P3; in AF-MSCs, only ITGB1, CD44, and ALCAM mRNAs are detected; NT5E is expressed from P2 and ENG has not been found at any passage. AF-MSCs and AECs are positive for the pluripotent markers (POU5F1 (OCT4) and MYC (c-Myc)) and lack of the hematopoietic markers. When appropriately induced, both cell lines are capable of differentiating into ectodermal and mesodermal lineages. This study contributes to reinforce the emerging importance of these cells as ideal tools in veterinary medicine. A deeper evaluation of the immunological properties needs to be performed in order to better understand their role in cellular therapy.

Fluorescent multiple staining and CASA system to assess boar sperm viability and membranes integrity in short and long-term extenders.

The aim of this study was to assess the effect on boar spermatozoa quality of in vitro storage in short and long-term extenders by fluorescent multiple staining (FMS) and computer assisted semen analyzer (CASA). Fresh ejaculates from three healthy, sexually mature boars were diluted with equal volumes of six short-term or three long-term commercial extenders and stored at 19°C for 6 days (short-term) or 12 days (long-term). The integrity of spermatozoa membranes was analyzed by FMS using propidium iodide, 5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) and fluorescein isothiocyanate-conjugated peanut agglutinin (PNA). The results obtained from this staining were compared with spermatozoa motility assessed by CASA. Our study showed that the number of viable spermatozoa with non-reacted acrosomes and intact mitochondria was positively correlated with the rate of motile spermatozoa (r(2)>0.9) irrespective of the extender used. In all extenders the number of motile spermatozoa significantly decreased as preservation period increased (P<0.05). FMS test is a potent indicator of sperm motility because it analyses mitochondrial integrity independently from observable alterations in motility. The best performing extenders were BTS for short-term storage and TRI-x-Cell for long-term storage.

Expression of aquaporin water channels in canine fetal adnexa in respect to the regulation of amniotic fluid production and absorption.

Amniotic fluid (AF) is created by the flow of fluid from the fetal lung and bladder and reabsorbed in part by fetal swallowing and partly by the transfer across the amnion to the fetal circulation. Placental water flux is an important factor in determining AF volume and fetal hydration. In addition the fetal membranes might be involved in the regulation of fluid composition. To understand the mechanisms responsible for maintaining a correct balance of AF volume we evaluated the expression of aquaporins (AQPs) in canine fetal adnexa. AQPs are a family of integral membrane proteins permitting passive but physiologically rapid transcellular water movement. The presence of AQP1, 3, 5, 8 and -9 was immunohistochemically assessed in canine fetal adnexa, collected in early, middle and late-gestation during ovario-hysterectomies performed with fully informed owners' consent. Changes in AF volume and biochemical composition were also evaluated throughout pregnancy. Our results show distinct aquaporin expression patterns in maternal and extraembryonic tissues in relation to pregnancy period. AQP1 was localized in placental endothelia, allantochorion, amnion, allantois and yolk sac. AQP3 was present in the placental labyrinth, amnion, allantois and yolk sac. AQP8 was especially evident on the epithelia lining the glandular chambers, the amniotic and allantois sacs. AQP9, a channel highly permeable to water and urea, was observed in epithelia of amnion, allantois and yolk sac. In summary, AQP1, 3, 5, 8 and -9 have distinct expression patterns in canine fetal membranes and placenta in relation to pregnancy period, suggesting an involvement in mediating the AF changes during gestation.

Characterization and potential applications of progenitor-like cells isolated from horse amniotic membrane.

The aim of this work was to isolate, for the first time, progenitor-like cells from the epithelial (AECs) and mesenchymal (AMCs) portions of the horse amniotic membrane, and to define the biological properties of these cells. AECs displayed polygonal epithelial morphology, while AMCs were fibroblast-like. Usually, six to eight passages were reached before proliferation decreased, with 13.08 and 26.5 cell population doublings attained after 31 days for AECs and AMCs, respectively. Immunocytochemical studies performed at passage 3 (P3) showed that both cell populations were positive for the expression of specific embryonic markers (TRA-1-60, SSEA-3, SSEA-4 and Oct-4). Meanwhile, RT-PCR performed at P1 and P5 showed expression of mesenchymal stem/stromal cell markers (CD29, CD105, CD44 and CD166) with negativity for CD34 at P1, although this marker began to be expressed by P5. The cells also expressed MHC-I at both P1 and P5, but lacked MHC-II expression at P1. Both AECs and AMCs demonstrated high plasticity, differentiating in vitro toward the osteogenic, adipogenic, chondrogenic and neurogenic lineages. Equine amnion-\derived cells could also be frozen and recovered without loss of their functional integrity in terms of morphology, presence of specific stemness markers and differentiation ability, although the renewal capacity was lower than that observed for freshly isolated cells. To investigate potential therapeutic effects and cell tolerance in vivo, horse amnion-derived cells were allogeneically injected into three horses with tendon injuries, resulting in a quick reduction in tendon size and ultrasonographic cross-sectional area measurements. These results suggest that horse amnion-derived cells may be useful for cell therapy applications.

Characterization and differentiation of equine tendon-derived progenitor cells.

Mesenchymal stem cells have been recently investigated for their potential use in regenerative medicine. Population of adult stem cells were recently identified in human and lab animal tendons, but no detailed investigations have been made in the equine species. The aim of our study is to identify a progenitor cell population from tendon tissue (TSPCs) in the horse superficial digital flexor tendon that are able to be highly clonogenic, to grow fast and to differentiate in different induced cell lineages as well as bone marrow derived progenitor cells (BM-MSCs). The hypothesis that TSPCs possess a mesenchymal stem cell behavior opens a new prospective for tendon regenerative medicine approaches. TSPCs were expanded more rapidly and showed higher plating efficiency when compared with BM-MSCs. Both cell lines expressed identical stem cell markers in vitro and they were able to differentiate towards osteogenic and adipogenic lineages as demonstrated with cytochemical staining and mRNA gene expression. TSPCs showed a positive but limited chondrogenic differentiation compared with BM-MSCs as demonstrated by histological and biochemical analyses. According to our results, equine TSPCs have high clonogenic properties and proliferating potential, they express stem cell markers and have the capability to be multipotent as well as BM-MSCs. These findings suggest that TSPCs may represent a good model for stem cell biology and could be useful for future tendon regenerative medicine investigations.

Size-sieved subpopulations of mesenchymal stem cells from intervascular and perivascular equine umbilical cord matrix.

OBJECTIVES: Umbilical cord matrix (UCM) has been recently proposed as an alternative source of mesenchymal stem cells (MSCs). The aim of this study was to isolate and characterize presumptive stem cells from intervascular and perivascular equine UCM and to obtain homogeneous subpopulations from both sites. MATERIALS AND METHODS: Umbilical cords were processed for retrieval of MSCs. Unsieved cells from intervascular and perivascular portions were evaluated for cell cycle analysis and for immunophenotyping by flow cytometry. Cells from each site were separated into larger and smaller sieved populations using multi-dishes with 8-µm pore transwell inserts. Each cell population was characterized in terms of renewal capability, specific marker expression and differentiation potential. Cryopreservation was performed on sieved cells only. RESULTS: Cells from both areas expressed MSC and pluripotential specific markers and were able to differentiate into mesodermic and ectodermic lineages. The sieving procedure yielded two relatively homogeneous subpopulations with comparable characteristics. Surprisingly, after sieving, large intervascular and small perivascular cells were the most rapidly replicating cells [20.53 and 19.49 cell population doublings (PD) after 31 days respectively] and also showed higher fibroblast colony forming unit frequency. Unsieved cell populations were used as controls, and showed PD of 9.42(intervascular cells) and 8.54 (perivascular cells) after 31 days. CONCLUSIONS: Here, cells from UCM represented an intermediate stage between pluripotent embryonic and adult stem cells. Size-sieving can be used to isolate more rapidly proliferating cell populations.

Isolation, proliferation, cytogenetic, and molecular characterization and in vitro differentiation potency of canine stem cells from foetal adnexa: a comparative study of amniotic fluid, amnion, and umbilical cord matrix.

The possibility to isolate canine mesenchymal stem cells (MSCs) from foetal adnexa is interesting since several canine genetic disorders are reported to resemble similar dysfunctions in humans. In this study, we successfully isolated, cytogenetically and molecularly characterized, and followed the differentiation potency of canine MSCs from foetal adnexa, such as amniotic fluid (AF), amniotic membrane (AM), and umbilical cord matrix (UCM). In the three types of cell lines, the morphology of proliferating cells typically appeared fibroblast-like, and the population doubling time (DT) significantly increased with passage number. For AF- and AM-MSCs, cell viability did not change with passages. In UCM-MSCs, cell viability remained at approximately constant levels up to P6 and significantly decreased from P7 (P < 0.05). Amnion and UCM-MSCs expressed embryonic and MSC markers, such as Oct-4 CD44, CD184, and CD29, whereas AF-MSCs expressed Oct-4, CD44. Expression of the hematopoietic markers CD34 and CD45 was not found. Dog leucocyte antigens (DLA-DRA1 and DLA-79) were expressed only in AF-MSCs at P1. Isolated cells of the three cell lines at P3 showed multipotent capacity, and differentiated in vitro into neurocyte, adipocyte, osteocyte, and chondrocyte, as demonstrated by specific stains and expression of molecular markers. Cells at P4 showed normal chromosomal number, structure, and telomerase activity. These results demonstrate that, in dog, MSCs can be successfully isolated from foetal adnexa and grown in vitro. Their proven stemness and chromosomal stability indicated that MSCs could be used as a model to study stem cell biology and have an application in therapeutic programs.

Fetal adnexa derived stem cells from domestic animal: progress and perspectives.

The fetal adnexa such as umbilical cord, amnion and amniotic fluid have been proposed as ideal sources of different stem cell lineages. Use of adnexal tissue has many potential advantages, including the noninvasive nature of the isolation procedure, the large tissue mass from which cells can be harvested with high efficiency and the potential of these cells to differentiate. Moreover, particularly in human medicine, the harvesting of these tissues is more ethically acceptable making these sources of stem cells very attractive for regenerative therapies and biotechnological applications. The adnexal tissue cells preserve some of the characteristics of the primitive embryonic layers from which they originate. Indeed, many studies indicate that these stem cells exhibit some features of embryonic stem cells as expression of embryonic markers and proliferation capability, without showing immunogenicity. However, the differentiation potential of these cells, either in vivo or in vitro, is intermediate between the pluripotent embryonic stem cells and the multipotent adult stem cells. Non-embryonic extra-fetal derived stem cells have opened new perspectives for developmental biology and for regenerative medicine, not only in humans but also in animals. In this update, we report the state of the art of fetal adnexa-derived stem cells from domestic animals and analyze their applications and potential uses in veterinary medicine.

Morphometric characteristics and chromatin integrity of spermatozoa in three Italian dog breeds.

OBJECTIVES: Studies in many species indicate that variation of spermatozoan head morphology is a sensitive biomarker for abnormal chromatin structure and resultant clinical fertility. This preliminary study evaluated spermatozoan head morphometry in different dog breeds and assessed whether morphometric parameters could reflect spermatozoan DNA fragmentation in dogs. METHODS: Spermatozoan morphometry and DNA quality (measured by TUNEL flow cytometry) were assessed in semen from 11 dogs of three Italian breeds (Cirneco dell'Etna, Piccolo Levriero Italiano and Segugio Maremmano). RESULTS: Morphometric data showed that Segugio dogs had significantly larger (33·67%) spermatozoa and that Piccolo Levrieros had a higher incidence of long (46·75%) and elliptical spermatozoan heads (11·5%) when compared with the samples from other breeds. Moreover, the predominance of elliptical spermatozoa in one dog (23%) was significantly related to the percentage of spermatozoa with fragmented DNA (12·6%), whereas in another dog, where no more than 1% of spermatozoa was elliptical, only 0·36% of spermatozoa had damaged DNA. It is noteworthy that the breeding record of the former dog in the previous 12 months showed poor fertility and fecundity. CLINICAL SIGNIFICANCE: These data suggest that spermatozoan head morphometry could be breed related and that there is a significant correlation between DNA fragmentation and elliptical spermatozoa in individual animals. This finding, albeit limited in our study to a single case, is possibly related to clinical infertility.

Efficacy of tuohy needle in oocytes collection from excised mare ovaries.

TWO METHODS HAVE BEEN DESCRIBED TO RECOVER OOCYTES FROM EQUINE FOLLICLES IN EXCISED OVARIES: aspiration and scraping. Aim of this work was to develop an effective method for collecting equine oocytes using Tuohy needle and comparing this technique to aspiration and scraping, with or without tunica albuginea removal. This hollow hypodermic needle, usually employed for inserting epidural catheters, is designed with a slightly curved tip, shaped similar to a small curette. In unpeeled ovaries, the recovery rates of Tuohy needle group was higher (P < .05) than in the 16 g needle aspiration and in the scraped ovaries (57% versus 36% and 47%) while the rate of cumulus-intact oocytes was higher than aspiration (46.9% versus 39.36%) but lower than scraping (46.97%) (P < .001). In unpeeled ovaries there was significant difference in maturation rate of oocytes recovered by Tuohy needle in respect to peeled ovaries (58.54% versus 50.17%, resp.). Combination of aspiration and scraping by Tuohy needle allows a faster and reliable collection of oocytes suitable for horse IVM.

Evaluation of newborn canine viability by means of umbilical vein lactate measurement, apgar score and uterine tocodynamometry.

Newborn viability evaluation and early detection of fetal distress could contribute to reducing mortality at birth in canine species. High neonatal mortality rate in dogs is reported subsequent to complicated or uncomplicated whelping. Umbilical vein lactate and tocodynamometry could provide valuable clinical information to the obstetricians so that appropriate medical and surgical treatments or oxygen and warm administration can be properly and timely applied to mother and newborn pup. In humans, the fetal lactate level represents an objective indicator of fetal distress and a valid predictor of babies' survival. Fetal acidosis recognition by umbilical lactate (UL) measurement, APGAR score classification, and uterine activity monitoring during labour, can represent an advanced system in the evaluation of the canine newborn patient. The purpose of this study was to correlate UL levels with canine neonatal morbidity and mortality within 48 h of birth. We evaluated the relationship among neonatal parameters at birth (mucous membrane color, heart and respiratory rate, reflex irritability, mobility, suckling and vocalization, UL, weight, and temperature) with labour characteristics (uterine contractions recorded by the tocodynamometric system of Whelpwise ™ Veterinary Perinatal Specialties ®, delivery time, and pup presentation), in view to predict pup viability. We considered also vaginal parturition versus elective and emergency Caesarean section, and uterotonic drugs influence on delivery. Umbilical lactate concentration proved to be useful to predict canine neonatal mortality within 48 h of birth (P < 0.05). We identified 5 mmol/L of vein umbilical lactate concentration as the cut off value, allowing us to distinguish between healthy and distressed pups. Higher values of UL were related with distressed pups, whereas lower values characterized vigorous pups. Lactate concentrations lower than 5 mmol/L and APGAR scores higher than 9, related to mean delivery time of 105 min with effective uterine contractions (10 mm of Hg of strength or more, frequency from 4 to 12 contractions per hour, and 2-5 min in duration), should be considered good prognostic factors in canine labour and neonatology.

An insight into testis and gubernaculum dynamics of INSL3-RXFP2 signalling during testicular descent in the dog.

Insulin-like 3 (INSL3) plays a prominent role in male development and is supposed to induce the growth of the gubernaculum testis (g.t.), thus being directly involved in testicular descent in humans and rodents. This happens through activation of the RXFP2 receptor (GREAT or LGR8). The INSL3-RXFP2 complex is reputed to play an additional paracrine role in the testis, possibly acting as part of an autocrine feedback loop. The present work provides evidence of the immunolocalisation of INSL3 in the Leydig cells of canine fetuses and of the expression of RXFP2 receptor in different tissues of the g.t. of the same specimens. RXFP2 was localised at the cell membrane of g.t. muscle and connective cells, as well as in the epithelial cells of the developing excurrent ducts. Notably, RXFP2 immunoreactivity of the g.t. was limited to fetuses at ~35-45 days of gestation, which is also the fetal period when the endocrine compartment of the dog testis is active endocrinologically, as confirmed by the anti-P450c17 and anti-INSL3 immunoreactivities of the fetal Leydig cells, and by anti-Müllerian hormone immunoreactivity of the Sertoli cells. The same immunoreactivities were also evaluated in the testes of cryptorchid dogs of different ages. RXFP2 immunoreactivity was absent from genital tracts of cryptorchid testes and g.t. remnants.

Endometrial cytology and computerized morphometric analysis of epithelial nuclei: a useful tool for reproductive diagnosis in the bitch.

New diagnostic approaches are required to recognize early canine hypofertility or infertility. We suggest that the identification of different cytologic types, cellular aspects, and nuclear features of the endometrial epithelial cells may be suitable for this purpose. This study was performed on the bitch (Canis familiaris) during the physiologic reproductive cycle and in uterine diseases. We also applied computerized cytomorphometry to evaluate nuclear area, perimeter, diameter, density, aspect, and roundness of endometrial epithelial cells in healthy dogs (N=35) at different stages of the reproductive cycle (before puberty, during proestrus, estrus, diestrus, and anestrus) and in bitches affected by uterine disorders (N=10). The stage of the estrous cycle was determined by vaginal cytology and progesterone evaluation and also confirmed by clinical and histologic observations. Samples for endometrial cytology were collected in vivo by uterine flushing with transcervical uterine cannulation. After uterine sampling, each dog underwent OHE or uterine stump revision. Cytologic analyses were compared with histologic examinations to verify the uterine condition. The uterine cellular population was represented by endometrial epithelial cells, erythrocytes, neutrophils, lymphocytes, eosinophils, macrophages, plasma cells, and cervical or incidental vaginal cells. Bacteria and amorphous material were observed. The proportion of different cells and nuclear features in the cytologic samples varied throughout the stages of the reproductive cycle and between normal and pathologic uterine conditions. The computer-assisted nuclear morphometry, performed in cytologic specimens by means of the six nuclear parameters chosen to evaluate the endometrial epithelial cell population, proved to be useful for determining the stage of the reproductive cycle. Furthermore, this system was demonstrated to be a valid support to diagnose and distinguish uterine disorders.

Time course of in vitro maturation of compact cumulus horse oocytes after roscovitine-induced meiotic inhibition: effects on the coordination between nuclear and cytoplasmic maturation.

This study was carried out to evaluate the usefulness of a pre-maturation step in improving the coordination between cytoplasmic and nuclear maturation of horse compact cumulus oocytes by the addition of roscovitine (ROSC). Oocytes were collected by scraping and pre-cultured for 18 h in a maturation medium TCM199 supplemented with pyruvate, LH, FSH, insulin growth factor (IGF), epidermal growth factor (EGF), insulin, transferrin and selenium (IVM-ROSC) or in a simple medium (M199-ROSC). After pre-maturation, oocytes from both the groups were in part denuded and fixed-stained and in part in vitro matured to assess the kinetic of in vitro maturation (IVM). The nuclear progression and the cytoskeletal organization of microfilaments and cortical granules (CG) of treated and untreated oocytes were assessed by fluorescent probes. Oocytes immediately fixed after recovery and oocytes pre-cultured in M199-ROSC for 18 h did not show metaphase II (MII) plates, whereas in IVM-ROSC group, 6/69 oocytes (8.7%) showed MII plates. After inhibition, during maturation kinetics at 11, 18 and 29 h, maturation rate of M199-ROSC group progressively increased and at 29 h of IVM, reached the maturation rate of control group (13/66, 19.7% vs 31/125, 24.8%). No statistically significant differences in cytoplasmic maturation were found. The number of MII plates after 29 h of IVM, was significantly higher (p < 0.05) in IVM-ROSC group (34/90) compared with M199-ROSC (13/66) and control groups (31/125) as well as the number of oocytes with microfilaments and CG distributed in cortical region (25/34 vs 3/13 and 7/31 respectively). Our results showed that pre-culturing in the presence of Roscovitine in a fully supplemented maturation medium containing gonadotropins and growth factors partially suppressed the meiotic maturation, but established a more suitable environment for improving cytoplasmic maturation of horse compact cumulus oocytes as defined by microfilaments and CG configuration.