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

Platelet Rich Plasma for Regenerative Medicine Treatment of Bovine Ovarian Hypofunction

Recent studies on cull cows have shown that ovarian abnormalities, particularly ovarian insufficiency, are the main cause of reproductive failure. The aim of this study was to treat bovine ovarian failure with intraovarian administration of autologous platelet rich plasma (PRP), which is rich in growth factors, chemokines, and cytokines that could stimulate follicular growth and steroidogenesis. Twelve cows with ovarian hypofunction were enrolled in the study and they were randomly allocated in control group (CTR) and treated group (six animal for group). In the treated group, only five animals received the PRP treatment because intraovarian administration was hindered in one by a rectovaginal fistula. Animals of control group were treated by intraovarian administration of physiological solution. In the 4 weeks after PRP injection, a mild to strong increase in progesterone (PRG) concentrations was detected in four of the five cows treated. Artificial insemination (AI) resulted in four pregnancies that are still ongoing (7th month). Intraovarian administration of PRP improved ovarian function after 2 months of treatment. This effect may be due to reduction of follicular atresia or to revitalization of dormant oocytes allowing restoration of fertility

Amniotic microvesicles impact hatching and pregnancy percentages of in vitro bovine embryos and blastocyst microRNA expression versus in vivo controls

Embryo development and implantation are dynamic processes, responsive to external signals, and can potentially be influenced by many environmental factors. The aims of this study were to evaluate the effects of a culture medium supplemented with amniotic-derived microvesicles (MVs) on in vitro embryo hatching after cryopreservation, and pregnancy rate following embryo transfer. In addition, miRNA profiling of blastocysts produced in vitro, with or without (control; CTR) amniotic MV supplementation, was also evaluated using blastocysts produced in vivo. In vitro embryos were cultured with and without amniotic MV supplementation. In vivo blastocysts were obtained from superovulated cows. Samples for RNA isolation were obtained from three pools of 10 embryos each (in vivo, in vitro-CTR and in vitro + MVs). Our results show that the hatching percentage of cryopreserved in vitro + MVs embryos is higher (P &lt; 0.05) than in vitro-CTR embryos and the pregnancy rate with fresh and cryopreserved in vitro + MVs embryos is higher than in vitro-CTR embryos. In addition, the analysis of differently expressed (DE) microRNAs showed that embryos produced in vivo are clearly different from those produced in vitro. Moreover, in vitro-CTR and in vitro + MVs embryos differ significantly for expression of two miRNAs that were found in higher concentrations in in vitro-CTR embryos. Interestingly, these two miRNAs were also reported in degenerated bovine embryos compared to good quality blastocysts. In conclusion, MV addition during in vitro production of embryos seems to counteract the adverse effect of in vitro culture and partially modulate the expression of specific miRNAs involved in successful embryo implantation

Antimicrobial Effects of Conditioned Medium From Amniotic Progenitor Cells in vitro and in vivo : toward Tissue Regenerative Therapies for Bovine Mastitis

There is increasing evidence to suggest that, in addition to their regenerative effect, mesenchymal stromal cells (MSCs), and their secretome have an anti-inflammatory and antimicrobial role in the innate immune response in conditions such as sepsis. However, there is no published information on the effect of MSCs in bovine mastitis. Mastitis often results in extensive tissue damage due to multi-microorganism co-infection. This study investigated the ability of amniotic-derived conditioned medium (CM), in vitro and in vivo, to counteract microbial action and restore healthy tissue capable of milk production. Following determination of a dose\u2013response curve, 10,000 colony-forming units (CFU) of Staphylococcus aureus (S. aureus) were inoculated into bovine mammary epithelial cell culture with and without 10% CM (supplemented either at the time of bacteria inoculation or after 4 h). Acridine orange staining was used to assess cell viability/apoptosis. Additionally, an in vivo study was performed using 48 dairy cows with acute and chronic mastitis, treated with CM (treated group) or antibiotics (control group). In vitro results showed that CM can attenuate bacterial growth, as evaluated by the number of CFU. After 24 h of culture with S. aureus, 89.67% of mammary epithelial cells treated with CM were still alive, whereas all cells cultured without CM were dead. Rates of epithelial cell survival (60.67%) were similar when CM was added 4 h after bacteria inoculation. There was no difference in somatic cell count between cases of acute mastitis in the CM-treated or control group in the in vivo study. However, relapses in chronic mastitis were less common in the group receiving CM. Our results show that CM is able to mitigate bacterial growth in vitro and may be particularly useful in the treatment of chronic mastitis, aiding restoration of milk production in cows that would otherwise be removed from the production cycle

Seasonal effects on miRNA and transcriptomic profile of oocytes and follicular cells in buffalo (Bubalus bubalis)

Season clearly influences oocyte competence in buffalo (Bubalus bubalis); however, changes in the oocyte molecular status in relation to season are poorly understood. This study characterizes the microRNA (miRNA) and transcriptomic profiles of oocytes (OOs) and corresponding follicular cells (FCs) from buffalo ovaries collected in the breeding (BS) and non-breeding (NBS) seasons. In the BS, cleavage and blastocyst rates are significantly higher compared to NBS. Thirteen miRNAs and two mRNAs showed differential expression (DE) in FCs between BS and NBS. DE-miRNAs target gene analysis uncovered pathways associated with transforming growth factor \u3b2 (TGF\u3b2) and circadian clock photoperiod. Oocytes cluster in function of season for their miRNA content, showing 13 DE-miRNAs between BS and NBS. Between the two seasons, 22 differentially expressed genes were also observed. Gene Ontology (GO) analysis of miRNA target genes and differentially expressed genes (DEGs) in OOs highlights pathways related to triglyceride and sterol biosynthesis and storage. Co-expression analysis of miRNAs and mRNAs revealed a positive correlation between miR-296-3p and genes related to metabolism and hormone regulation. In conclusion, season significantly affects female fertility in buffalo and impacts on oocyte transcriptomic of genes related to folliculogenesis and acquisition of oocyte competence

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

Secretome of bovine amniotic and endometrial cells : application for in vitro embryo production

Some maternal miRNAs are involved in early stage embryos [Abd El Naby, 2011]. Microvesicles (MVs) have been suggested as carrier of miRNAs for maternal - to - embryonic communication during the first days of early development [Mondou, 2012]. MVs, together with soluble factors, are components of conditioned media (CM) produced by cells during their culture. Aim of this study was to understand the role of CM, MVs and supernatant (SN, obtained from CM deprived of MVs) secreted by bovine endometrial and amnion cells on embryo development. In vitro produced embryos were cultured in SOFaa alone (CTR) or su pplemented on day 5 post-fertilization with 10% of endometrial or amniotic CM or SN or 100x106 MVs/ml. The blastocyst rate obtained culturing embryos with amniotic CM and MVs was not significantly different from the CTR (34.17\ub13.29%, 32.82\ub13.26% and 35.45\ub1 2.53% respectively). The rate obtained by amniotic SN was 25.80\ub12.83% and statistically lower (P<0.05) than the other groups. The rate obtained by endometrial products were lower than CTR and the other conditions. The ICM of embryos cultured in medium supplemented with amniotic components had a higher number of cells than the CTR group: 30.4\ub11.83 and 29.42\ub11.27 for CM and MVs respectively compared to 27.6\ub11.44 for CTR (P<0.05). Our data showed that exposing the embryos to the amniotic secretome does not imp rove the blastocyst rate, but increases their quality. The hypothesis is that miRNAs contained into MVs may contribute to the production of better quality embryos and that amniotic secretome supplies a more physiological environment for the embryo development

Evaluation of amniotic mesenchymal cell derivatives on cytokine production in equine alveolar macrophages : an in vitro approach to lung inflammation

Background: Data obtained in both animal models and clinical trials suggest that cell-based therapies represent a potential therapeutic strategy for lung repair and remodeling. Recently, new therapeutic approaches based on the use of stem cell derivatives (e.g., conditioned medium (CM) and microvesicles (MVs)) to regenerate tissues and improve their functions were proposed. The aim of this study was to investigate the immunomodulatory effects of equine amniotic mesenchymal cell derivatives on lipopolysaccharide (LPS)-induced cytokine production in equine alveolar macrophages, which may be beneficial in lung inflammatory disorders such as recurrent airway obstruction (RAO) in horses. RAO shares many features with human asthma, including an increased number of cells expressing mRNA for interleukin (IL)-4 and IL-5 and a decreased expression of IFN-\u3b3 in bronchoalveolar lavage fluid (BALF) of affected horses. Methods: The release of TNF-\u3b1, IL-6, and TGF-\u3b21 at different time points (1, 24, 48, and 72 h) was measured in equine alveolar macrophages stimulated or not with LPS (10 and 100 ng/mL) in the presence or absence of 10 % CM or 50 7 106 MVs/mL. Cytokines were measured using commercially available ELISA kits. For multiple comparisons, analysis of variance was used with Tukey post-hoc test. Differences were considered significant at p 64 0.05. Results: Significant modulatory effects of CM on LPS-induced TNF-\u3b1 release at 24 h, and of both CM and MVs on TNF-\u3b1 release at 48 h were observed. A trend toward a modulatory effect of both CM and MVs on the release of TGF-\u3b2 and possibly IL-6 was visible over time. Conclusions: Results support the potential use of CM and MVs in lung regenerative medicine, especially in situations in which TGF-\u3b2 may be detrimental, such as respiratory allergy. Further studies should evaluate the potential clinical applications of CM and MVs in equine lung diseases, such as RAO and other inflammatory disorders

Effect of lipid peroxidation on the immunocytochemical detection of a leukocyte antigenic determinant in fresh and cryopreserved bovine spermatozoa

Studies on different species, including rats, monkeys and humans, have shown the presence of leukocyte differentiation antigens in the spermatozoa. In some case the expression of these molecules is related to a specific functional state of the sperm cell, as was found for the CD 46 antigen, that in humans can be used as a marker of the acrosome reaction. The aim of the present study was to assess wether promoted lipid peroxidation of the spermatozoa induces any variations in their immunoreactivity with ILA 147 antibody that, in bull spermatozoa, recognizes bovine leukocyte antigens. Freshly ejaculated bovine spermatozoa and cryopreserved semen were tested for ILA 147 reactivity by standard immunoperoxidase staining, before and after promoted lipid peroxidation. Staining intensity was assessed in the individual cells using the microdensitometric method to measure integrated optical density (IOD), overcoming the disadvantage of an operator's subjective interpretation of the results. After the lipid peroxidation there was significantly decreased staining intensity in the fresh spermatozoa, but not in the cryopreserved cells. Furthermore, in the preincubation conditions, the cryopreserved spermatozoa had a lower mean I.O.D. value than the fresh sperm, showing that the freezing and thawing processes induced an alteration in the antigen exposure. However the mean immunoreactivity of the cryopreserved cells was not significantly influenced by lipid peroxidation. The absorbance value maps, made following immunoperoxidase staining by the examined antibody, showed that the reaction sites in the fresh and cryopreserved spermatozoa fell mainly within the periacrosomal region. Moreover, after induced lipid peroxidation there were fewer reaction sites in this domain. The present research has confirmed the presence of the examined leukocyte antigenic determinant in the bull spermatozoa, and suggests that promoted lipid peroxidation and the freezing and thawing of spermatozoa can produce membrane damage, leading to reduced ILA 147 antigenic site exposure