Embryo transfer and related technologies in sheep reproduction

This paper reviews the status of embryo transfer and the major technologies applied to preimplantation of embryos in sheep. Embryo production from superovulated ewes is hindered by an unpredictable response to hormonal treatment. Progress in this area should be expected by an appropriated control of follicular development with gonadotropin-releasing hormone (GnRH) agonist or antagonist prior to gonadotrophin administration. Simple protocols for the cryopreservation of sheep embryos by vitrification are already available and the development of frozen-thawed blastocysts to term is close to the fresh ones. Further research is required to identify factors able to promote the maturation in vitro of oocytes, namely those obtained from prepubertal animals. Semen and embryo sexing procedures are available in cattle although much less attention was paid to their application to sheep. Among all the reproductive technologies, cloning with embryonic and foetal cells has progressed dramatically in sheep and nuclear transfer has been used to produce transgenic animals as an alternative to pronuclear injection. The production of the first lamb cloned from a somatic cell opened new opportunities in animal breeding as well as exciting lines of basic research. The overall conclusions are that, apart from superovulation, the application of in vitro technologies is likely to evolve rapidly and once applied, a great impact on traditional and new animal productions should be expected. However, a better understanding of the changes in gene expression, induced in embryos by different in vitro manipulation procedures, is necessary to prevent abnormal foetal development. Cette revue traite du transfert d’embryons et des principales biotechnologies appliquées à l’embryon ovin. La production d’embryons, après superovulation, est limitée par la réponse non reproductible au traitement hormonal. Un progrès pourrait venir d’un contrôle approprié du développement folliculaire avec un agoniste ou antagoniste de GnRH appliqué avant le traitement gonadotrope. Des protocoles simples pour la congélation d’embryons ovins par vitrification sont disponibles et, après décongélation, le développement à terme de blastocystes congelés est proche de celui des embryons frais. De nouvelles recherches seront nécessaires pour identifier les facteurs capables de stimuler la maturation in vitro des ovocytes, en particulier ceux d’animaux prépubères. Le sexage des spermatozoïdes et des embryons est possible chez les bovins, mais peu appliqué chez les ovins. De toutes les techniques de reproduction, c’est celle du clonage à partir de cellules embryonnaires ou foetales qui a le plus progressé; le transfert nucléaire a été utilisé pour produire des animaux transgéniques, comme alternative à l’injection dans les pronoyaux. La production du premier agneau cloné à partir d’une cellule somatique a ouvert de nouvelles perspectives en élevage et en recherche fondamentale. En conclusion, excepté dans le domaine de la superovulation, les biotechnologies vont évoluer rapidement; leur application aura certainement un grand impact sur les méthodes traditionnelles et nouvelles de production. Cependant, une meilleure connaissance des effets sur l’expression de gènes embryonnaires induits par les manipulations in vitro serait nécessaire pour éviter un développement foetal anormal

Two culture systems showing a biphasic effect on ovine embryo development from the 1-2 cell stage to hatched blastocysts

This study compared the effect of using either CZB or TCM 199 media on both the development of 1-2 cell ovine embryos from superovulated ewes to the blastocyst stage (Experiment 1), and the hatching process of ovine blastocysts developed in vitro (Experiment 2). For the first 5 d, the CZB medium showed higher rates of embryo development than the TCM 199 medium (p < 0.001). The embryos reaching the > 16 cell stage were 79 vs 52% and 74 vs 20% with or without an oviductal monolayer, respectively, and those reaching the blastocyst stage were 71 vs 46% and 46 vs 13% with or without cells. The CZB medium was less able to support the hatching process of the blastocysts obtained in the first experiment than was the TCM-199 medium + 10% FCS (fetal calf serum) with cells (31 vs 92%; p < 0.001) or without cells (13 vs 66%; p < 0.001). No blastocysts completely escaped from the zona pellucida (ZP) in the CZB medium compared with 80 and 61 % in the TCM 199 medium with or without cells, respectively. In Experiment 3, 47% of the blastocysts migrated through the artificial opening of the ZP and hatched completely. After 24 h of culture in the CZB medium, however, they showed blastocoelic cavity breakdown. During the preliminary cleavages, the ovine embryos developed better in CZB medium than in TCM 199, but the latter was more efficient in promoting the hatching process of the blastocysts. Les effets d’un milieu de culture, CZB, et d’un milieu de culture, TCM 199 + 10% de sérum de veau foetal, ont été observés sur le développement in vitro d’embryons d’ovins du stade 1-2 cellules au stade blastocyste (expérience 1) et dans le processus d’éclosion des blastocystes produits lors de l’expérience précédente (expérience 2). Le milieu CZB exerce un effet positif sur le développement embryonnaire par rapport au milieu TCM 199 après culture jusqu au stade > 16 cellules (79 vs 52 % et 74 vs 20%) ou jusqu’au stade blastocyste (71 vs 46% et 46 vs 13%) en présence de cellules et en l’absence de cellules tubaires respectivement. Le processus d’éclosion est plus faible dans le milieu CZB que dans le milieu TCM 199 soit en présence (31 vs 92%) soit en l’absence (13 vs 66%) de cellules tubaires respectivement. Dans le CZB (expérience 3), 47% des blastocystes éclosent par une fente artificielle dans la zone pellucide, mais, après 24 h de culture, la cavité blastocoelique se dégonfle. Ces résultats montrent que le milieu CZB est plus indiqué pour le développement précoce d’embryons ovins alors que le milieu TCM 199 est plus efficace au moment de la phase d’éclosion

Diffuse C-Cells Hyperplasia Is the Source of False Positive Calcitonin Measurement in FNA Washout Fluids of Thyroid Nodules: A Rational Clinical Approach to Avoiding Unnecessary Surgery

Purpose: The FNA-CT is useful for the diagnosis of MTC. The aim of this study was to evaluate the performance of FNA-CT in TNs coexisting with CCH. Methods: This study retrospectively reviewed the records of 11 patients with TNs submitted to thyroidectomy on the basis of elevated basal and/or stimulated serum CT values, which at histology were not confirmed to be MTC. The results obtained in this group were compared with those of a previously reported group of histologically proven MTC patients submitted to an identical presurgical evaluation. All patients, negative for known mutations in the RET proto-oncogene, were preoperatively submitted to neck ultrasound, FNA-cytology, and FNA-CT. Results: Approximately 6 of 11 patients showed increased (>36 ng/mL, as established in previous studies not involving patients with CCH) FNA-CT. All these patients showed diffuse CCH at histology in the thyroid lobe submitted to FNA; 5 of them were benign at histology, while only one was malignant (papillary thyroid carcinoma, PTC). The remaining 5 of 11 patients had low FNA-CT (<36 ng/mL), and all of them showed only focal CCH in the lobe submitted to FNA; three of them were malignant (2 PTC, 1 follicular carcinoma), while two were benign. Conclusions: Employing the currently proposed cut-off values, false-positive FNA-CT results may be observed in benign/malignant TNs with coexisting diffuse CCH. FNA-CT must therefore be cautiously used in the diagnostic approach for patients with TNs and a slightly increased basal or stimulated serum CT concentration in order to avoid unnecessary surgery

Comparison of laparoscopic and transcervical insemination with frozen semen in Sarda dairy ewes

Laparoscopic insemination with frozen-thawed semen is currently used for planned matings in the Sarda breeding programme. In order to find a fast and less intrusive artificial insemination (AI) method that could replace laparoscopic insemination, a field comparison of laparoscopic and transcervical techniques was carried out on 200 mature Sarda ewes. After AI, ewes were assigned to teaser and fertile rams for 2 months. Return rates and cumulative (AI + natural mating) lambing rates were recorded over three subsequent 23-day periods. Lambing rates to AI were significantly different (P < 0·01), and were 62% and 7% respectively for laparoscopic and transcervical AI. Cumulative lambing rates after two further 23-day periods of natural mating were no longer significantly different (P > 0·05) and reached 82% and 74% respectively. Ewes with body condition scores at AI higher than 2·75 showed better overall reproductive performance, but not higher pregnancy rate to AI. Plasma cortisol concentrations, sampled twice, before and after AI, were higher (P < 0·01) in the last sample, suggesting a stress response to insemination. Cortisol levels after AI were lower (P < 0·01) for ewes submitted to transcervical rather than laparoscopic insemination (P < 0·01). However, cortisol levels after AI were no greater than those recorded when ewes were restrained in a milking yoke different from that usually employed. Laparoscopic AI was confirmed as the most suitable technique for insemination offrozen semen in the Sarda breeding scheme

Changes in Estimating the Wild Boar Carcasses Sampling Effort: Applying the EFSA ASF Exit Strategy by Means of the WBC-Counter Tool

African swine fever (ASF) is a devastating disease, resulting in the high mortality of domestic and wild pigs, spreading quickly around the world. Ensuring the prevention and early detection of the disease is even more crucial given the absence of licensed vaccines. As suggested by the European Commission, those countries which intend to provide evidence of freedom need to speed up passive surveillance of their wild boar populations. If this kind of surveillance is wellregulated in domestic pig farms, the country-specific activities to be put in place for wild populations need to be set based on wild boar density, hunting bags, the environment, and financial resources. Following the indications of the official EFSA opinion 2021, a practical interpretation of the strategy was implemented based on the failure probabilities of wrongly declaring the freedom of an area even if the disease is still present but undetected. This work aimed at providing a valid, applicative example of an exit strategy based on two different approaches: the first uses the wild boar density to estimate the number of carcasses need to complete the exit strategy, while the second estimates it from the number of wild boar hunted and tested. A practical free access tool, named WBC-Counter, was developed to automatically calculate the number of needed carcasses. The practical example was developed using the ASF data from Sardinia (Italian island). Sardinia is ASF endemic from 43 years, but the last ASFV detection dates back to 2019. The island is under consideration for ASF eradication declaration. The subsequent results provide a practical example for other countries in approaching the EFSA exit strategy in the best choices for its on-field applicatio

ADP and other metabolites released from <i>Acanthamoeba castellanii</i> lead to human monocytic cell death through apoptosis and stimulate the secretion of proinflammatory cytokines

Monocytes/macrophages are thought to be involved in Acanthamoeba infections. The aim of this work was to study whether soluble metabolites (ADP and other compounds) released by Acanthamoeba castellanii trophozoites could induce morphological and biochemical changes in human monocytic cells in vitro. We demonstrate here that ADP constitutively released in the medium by A. castellanii, interacting with specific P2y2 purinoceptors expressed on the monocytic cell membrane, caused a biphasic rise in [Ca2+]i, morphological changes characteristics of cells undergoing apoptosis, caspase-3 activation, and secretion of tumor necrosis factor alpha (TNF-α). The same results were found in monocytes exposed to purified ADP. Cell damage and TNF-α release induced by amoebic ADP were blocked by the P2y2 inhibitor suramin. Other metabolites contained in amoebic cell-free supernatants, with molecular masses of, respectively, &gt;30 kDa and between 30 and 10 kDa, also caused morphological modifications and activation of intracellular caspase-3, characteristics of programmed cell death. Nevertheless, mechanisms by which these molecules trigger cell damage appeared to differ from that of ADP. In addition, other amoebic thermolable metabolites with molecular masses of &lt;10 kDa caused the secretion of interleukin-1β. These findings suggest that pathogenic free-living A. castellanii by release of ADP and other metabolites lead to human monocytic cell death through apoptosis and stimulate the secretion of proinflammatory cytokines

Isolation, culture, and characterization of embryonic cell lines from vitrified sheep blastocysts

This study was conducted to isolate, to culture, and to characterize embryonic cell lines from in vitro produced vitrified sheep blastocysts. Embryos were produced and vitrified at the expanded blastocyst stage. Ten inner cell masses arising from day 6-7 blastocysts were isolated by immunosurgery, disaggregated, and cultured onto mitomocin-C-inactivated mouse STO fibroblasts (MIF). After 5 or 6 days of culture the primary cell colonies were disaggregated, seeded in a new MIF, and cultured for 3 or 4 days to form new colonies called Passage 1. These cells were then disaggregated and cultured for other two passages. The primary cell colonies and Passage 2 colonies expressed stage specific embryonic markers SSEA-1, SSEA-3, and SSEA-4, and were alkaline phosphatase positive. In the absence of feeder layer and human leukemia inhibitory factor (LIF), these cells differentiated into variety of cell types and formed embryoid bodies. When cultured for an extended period of time, embryoid bodies differentiated into derivatives of three embryonic germ (EG) layers. These were characterized by detection of specific markers for differentiation such early mesoderm (FE-C6), embryonic myosin (F1-652), neural precursor (FORSE-1), and endoderm (anti-cytokeratin 18). To our knowledge, this is the first time that embryonic cell lines from in vitro produced and vitrified ovine blastocysts have been isolated and examined for detection of SSEA markers, and embryoid bodies have been cultured and examined for specific cell surface markers for differentiation

Sexing of in vitro produced ovine embryos by duplex PCR

The aim of this article was to develop a fast and easy duplex polymerase chain reaction (PCR) method, for sex determination of ovine in vitro produced embryos prior to implantation. We tested the approach with 107 samples of autosomal cells (oviductal sheep cells and male lamb fibroblasts), divided into three groups for each sex according to the number of cells employed (30, 5, 2, respectively). We then used the test on 21 embryos at blastocyst stage. On the same day the embryos were transferred in pairs into 11 recipient synchronized ewes. The PCR utilized two different sets of primers: the first pair recognized a bovine Y-chromosome-specific sequence (SRY), that showed 100% homology with the corresponding sequence of the ovine Y-chromosome and is amplified in males only. The second pair recognized the bovine 1.715 satellite DNA (SAT) which was amplified in all ovine samples but, when submitted to the GenBank database did not show homology with any of the reported ovine sequences. However, after sequencing, ovine amplification product showed 98% homology with the bovine specific satellite sequence. The autosomal samples were amplified with 85.0% efficiency and 91.2% accuracy, while amplification was successful with all 21 embryos (100% efficiency). Eight lambs were born and the sex as determined by PCR corresponded to the anatomical sex in seven (87.5% accuracy). These results confirm that this method can be applied in ovine breeding programs to manipulate sex ratio of offspring