Ovum Pick-up in cattle – a 25-yr retrospective analysis

Repeated oocyte collection by transvaginal ultrasound-guided follicular puncture (Ovum Pick-Up, OPU), implicitly associated to in vitro embryo production (IVEP), has become alternative and competitive to superovulation for embryo production in cattle. It is alternative because it can be applied successfully irrespective of the reproductive status of the donor, i.e. in pregnant and acyclic animals, in those having patent tube or genital tract infections and in animals insensitive to superovulatory treatment. It is competitive because it can yield more transferable embryos per donor on a monthly basis. Through the years, the number of transferable embryos provided by OPU has significantly increased mainly due to the technological improvement of IVEP. However, limits to OPU application remain due to lower pregnancy rate of in vitro vs in vivo produced embryos or non optimal co-operation between OPU practitioners and IVF laboratories. This review will focus on the technical modifications proposed for improving OPU efficiency, the analysis of the physiological parameters that affect OPU and IVEP efficiency and, finally, the use of OPU as a tool to study and manipulate reproductive activity in cattle

gamete quality in a multistressor environment

Over the past few decades, accumulated evidence confirms that the global environment conditions are changing rapidly. Urban industrialization, agriculture and globalization have generated water, air and soil pollution, giving rise to an environment with a growing number of stress factors, which has a serious impact on the fitness, reproduction and survival of living organisms. The issue raises considerable concern on biodiversity conservation, which is now at risk: it is estimated that a number of species will be extinct in the near future. Sexual reproduction is the process that allows the formation of a new individual and is underpinned by gamete quality defined as the ability of spermatozoa and oocytes to interact during fertilization leading to the creation and development of a normal embryo.This review aimed to provide the current state of knowledge regarding the impact of a broad spectrum of environmental stressors on diverse parameters used to estimate and evaluate gamete quality in humans and in canonical animal models used for experimental research.Effects of metals, biocides, herbicides, nanoparticles, plastics, temperature rise, ocean acidification, air pollution and lifestyle on the physiological parameters that underlie gamete fertilization competence are described supporting the concept that environmental stressors represent a serious hazard to gamete quality with reproductive disorders and living organism failure. Although clear evidence is still limited, gamete capacity to maintain and/or recover physiological conditions is recently demonstrated providing further clues about the plasticity of organisms and their tolerance to the pressures of pollution that may facilitate the reproduction and the persistence of species within the scenario of global change.Changes in the global environment must be urgently placed at the forefront of public attention, with a massive effort invested in further studies aimed towards implementing current knowledge and identifying new methodologies and markers to predict impairment of gamete quality. Keywords: Gamete quality, Environmental stress, Fertilization, Xenobiotic, Climate change, Life styl

Developmental Potential in Bovine Oocytes Is Related to Cumulus-Oocyte Complex Grade, Calcium Current Activity, and Calcium Stores

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Analytical Validation of Two Assays for Equine Ceruloplasmin Ferroxidase Activity Assessment

: Ceruloplasmin (Cp) assessment in biological samples exploits the oxidase activity of this enzyme against several substrates, such as p-phenylenediamine (p-P), o-dianisidine (o-D) and, most recently, ammonium iron(II) sulfate (AIS). Once developed in humans, these assays are often used in veterinary medicine without appropriately optimizing in the animal species of interest. In this study, two assays using AIS and o-D as substrates have been compared and validated for Cp oxidase activity assessment in horse's plasma. The optimization of the assays was performed mainly by varying the buffer pH as well as the buffer and the substrate molar concentration. Under the best analytical conditions obtained, the horse blood serum samples were treated with sodium azide, a potent Cp inhibitor. In the o-D assay, 500 µM sodium azide treatment completely inhibits the enzymatic activity of Cp, whereas, using the AIS assay, a residual analytical signal was still present even at the highest (2000 µM) sodium azide concentration. Even though the analytical values obtained from these methods are well correlated, the enzymatic activity values significantly differ when expressed in Units L-1. A disagreement between these assays has also been detected with the Bland-Altman plot, showing a progressive discrepancy between methods with increasing analytical values

Meiosis progression and donor age affect expression profile of DNA repair genes in bovine oocytes

Several genetic and physiological factors increase the risk of DNA damage in mammalian oocytes. Two critical events are meiosis progression, from maturation to fertilization, due to the extensive chromatin remodelling during genome decondensation and aging which is associated to a progressive oxidative stress. In this work, we studied the transcriptional patterns of three genes, RAD51, APEX-1 and MLH1, involved in DNA repair mechanisms. The analyses were performed by Real-Time quantitative PCR (RT-qPCR) in immature and in vitro matured oocytes collected from 17 ± 3 mo old heifers and 94 ± 20 mo old cows. Batches of 30-50 oocytes for each group (three replicates) were collected from ovarian follicles of slaughtered animals. The oocytes were freed from cumulus cells at the time of follicle removal, or after IVM carried out in M199 supplemented with 10% foetal calf serum, 10 IU LH /ml, 0.1 IU FSH /ml and 1 µg 17β-oestradiol/ml. Total RNA was extracted by Trizol method. The expression of bovine GAPDH gene was used as internal standard, while primers for bovine RAD51, APEX-1 and MLH1 genes were designed from DNA sequences retrieved from GeneBank. Results obtained indicate a clear up-regulation of RAD51, APEX-1 and MLH1 genes after IVM ranging between 2- and 4-fold compared to GV oocytes. However, only RAD51 showed a significant transcript increase between the immature oocytes collected from young and old individuals. This finding candidates RAD51 as gene marker for discriminating bovine immature oocytes in relation to the donor age

Effects of heat stress on reproductive activity in dairy cows bred in the Potenza district

The effect of heat stress on the reproductive performance in dairy cows reared in Apennines areas of Southern Italy was evaluated. Reproductive parameters obtained from three farms during the period 2007-2012 were related to either season variations or the temperature-humidity index (THI), i.e., a complex climate parameter obtained by the maximum temperature and minimum relative humidity. The THI was able to assess the HS effects on parameters as conception rate on an annual basis (CRY) (R=-0.437; P <0.01) but was less efficient for parameters as the conception rate (CR). Whereas (i) CRY is influenced by both heat detection rate (HDR) and CR; (ii) an indirect analysis detected a significant (P< 0.001) reduction in the HDR along with THI increase; and (iii) CR was only partially affected by either THI or season, it follows that the main cause of reduced fertility in the farms surveyed was the HDR. The number of days open was significantly larger in the animals calved from January to July than in those calved between August and December (163±33 vs 123±36; P< 0.001); this increase may be because of the rescue of reproduction activity in the cows calved during the former period coincides with heat stress occurrence

Fluorescence Spectroscopy for the Diagnosis of Endometritis in the Mare

By exploiting the PMN property to produce high quantities of oxygen peroxide to neutralize pathogens, the oxygen peroxide content of uterine cells was measured to diagnose endometritis. After preliminary in vitro studies in which endometrial cells from slaughtered mares were mixed with leukocytes from peripheral blood, endometrial samples were collected by uterine flushing from mares before insemination. Staining endometrial cells with H2DCF‐DA was combined with hydroethidine to normalize the fluorescence intensity with the cellular content of the sample. Stained cell smears were assumed as the gold standard of endometritis, and based on this assay, the samples were considered positive (C+) and negative (C−) for endometritis. The amount and the turbidity of fluid recovered by uterine flushing were significantly (p &lt; 0.01) higher in C+ than in C−. Moreover, the oxygen peroxide content of the endometrial cells was significantly higher in the C+ than in the C− group (6.31 ± 1.92 vs. 3.12 ± 1.26, p = 0.001). Using the value of 4.4 as the cutoff level of this fluorescence cytology assay, it was found that only one C− sample exceeded the cutoff level (false positives = 7.7%) while three C+ samples showed values below the cutoff level (false negative = 11.5%)

Relationships between Seminal Plasma Metabolites, Semen Characteristics and Sperm Kinetics in Donkey (Equus asinus)

This study aimed to evaluate donkey seminal plasma metabolites and relate this information to the main characteristics of sperm quality. Sperm kinetics from 10 donkey stallions were analyzed with a computerized system at the time of collection (T0) and after 24 h storage at 4 °C (T24). Seminal plasma was frozen at -80 °C for subsequent proton nuclear magnetic resonance (1H NMR) spectroscopy. On three stallions, semen collection was repeated monthly for three times and sperm analysis also included mitochondrial activity and oxidative status. One stallion was azoospermic and a second semen collection was performed after one month. In the seminal plasma, 17 metabolites were identified; their levels showed numerous significant variations between the azoospermic and the normospermic individuals and grouped in well-defined clusters in a multivariate analysis. Comparing individuals with high and low sperm motility, the only discriminating metabolite was phenylalanine, whose levels were lower in the latter, as in the azoospermic individual. Phenylalanine was also the only metabolite highly correlated with all sperm kinematic parameters at T24. In conclusion, the present study has provided relevant information on the chemical characteristics of donkey semen, identified relationships between seminal metabolites, semen parameters, and sperm kinetics, and offered insights for future technological applications