Reviving vacuum-dried encapsulated ram spermatozoa via ICSI after 2 years of storage.

Introduction: Freeze-drying techniques give alternative preservation mammalian spermatozoa without liquid nitrogen. However, most of the work has been conducted in the laboratory mouse, while little information has been gathered on large animals that could also benefit from this kind of storage.

Methods: This work adapted a technique known as vacuum-drying encapsulation (VDE), originally developed for nucleic acid conservation in anhydrous state, to ram spermatozoa, and compared it to canonical lyophilization (FD), testing long-term storage at room temperature (RT) and 4°C.

Controlled spermatozoa-oocyte interaction improves embryo quality in sheep.

The current protocols of in vitro fertilization and culture in sheep rely on paradigms established more than 25 years ago, where Metaphase II oocytes are co-incubated with capacitated spermatozoa overnight. While this approach maximizes the number of fertilized oocytes, on the other side it exposes them to high concentration of reactive oxygen species (ROS) generated by active and degenerating spermatozoa, and positively correlates with polyspermy. Here we set up to precisely define the time frame during which spermatozoa effectively penetrates and fertilizes the oocyte, in order to drastically reduce spermatozoa-oocyte interaction.

Whole genome integrity and enhanced developmental potential in ram freeze-dried spermatozoa at mild sub-zero temperature.

Freeze-dried spermatozoa typically shows a reduction in fertility primarily due to the DNA damage resulting from the sublimation process. In order to minimize the physical/mechanical damage resulting from lyophilization, here we focused on the freezing phase, comparing two cooling protocols: (i) rapid-freezing, where ram sperm sample is directly plunged into liquid nitrogen (LN-group), as currently done; (ii) slow-freezing, where the sample is progressively cooled to - 50 °C (SF-group). The spermatozoa dried in both conditions were analysed to assess residual water content by Thermal Gravimetric Analysis (TGA) and DNA integrity using Sperm Chromatin Structure Assay (SCSA).

The impaired development of sheep ICSI derived embryos is not related to centriole dysfunction.

While intracytoplasmic sperm injection (ICSI) is an asset in human Assisted Reproduction Technologies (ART), its outcomes, in terms of blastocyst, is still unacceptably low in ruminants. The picture typically found in ICSI derived bovine and ovine embryos is an asymmetry between a high activation rate, marked by a pronuclear development, and a low first cleavage rate. Abnormal centriole function has been indicated as a possible factor which undermines embryonic development following ICSI, especially when Freeze Dried spermatozoa (FD) are used.

Maternal peri-conceptional undernourishment perturbs offspring sperm methylome.

The genotype of an organism is stable throughout its life; however, its epigenome is dynamic and can be altered in response to environmental factors, such as diet. Inheritance of acquired epigenetic modifications by the next generation occurs through the germline, although the precise mechanisms remain to be elucidated. Here, we used a sheep model to evaluate if modification of the maternal diet (CTR; control, UND: undernutrition; FA: undernutrition and folic acid supplementation) during the peri-conceptional period affects the genome-wide methylation status of the gametes of male offspring.

Dry biobanking as a conservation tool in the Anthropocene.

Species are going extinct at an alarming rate, termed by some as the sixth mass extinction event in the history of Earth. Many are the causes for this but in the end, all converge to one entity - humans. Since we are the cause, we also hold the key to making the change.

Somatic cell nuclear transfer: failures, successes and the challenges ahead.

Somatic cell nuclear transfer (SCNT) has a broad spectrum of potential applications, including rescue of endangered species, production of transgenic animals, drug production, and regenerative medicine. Unfortunately, the efficiency of SCNT is still disappointingly low. Many factors affecting cloning procedures have been described in several previous reviews; here we review the most effective improvements in SCNT, with a special emphasis on the effect of mitochondrial defects on SCNT embryo/ foetus development, an issue never touched upon before.

DNA fragmentation in epididymal freeze-dried ram spermatozoa impairs embryo development.

Sperm freeze-drying is a revolutionary technique, which has been gaining prominence in recent years. The first related significant result was Wakayama and Yanagimachi's demonstration in 1998 of the birth of healthy mouse offspring by Intracytoplasmic Sperm Injection (ICSI), using epididymal freeze-dried spermatozoa. Mouse, rat, and hamster models were the first small mammals born from lyophilized epididymal spermatozoa, whereas most other studies in this field used ejaculated spermatozoa.

Freeze-dried spermatozoa: An alternative biobanking option for endangered species.

In addition to the iconic wild species, such as the pandas and Siberian tigers, an ever-increasing number of domestic species are also threatened with extinction. Biobanking of spermatozoa could preserve genetic heritages of extinct species, and maintain biodiversity of existing species. Because lyophilized spermatozoa retain fertilizing capacity, the aim was to assess whether freeze-dried spermatozoa are an alternative option to save endangered sheep breeds.

Polychlorinated biphenyls (PCBs) alter DNA methylation and genomic integrity of sheep fetal cells in a simplified in vitro model of pregnancy exposure.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants ubiquitously detectable in the environment and in the food chain. Prenatal exposure to PCBs negatively affects fetal development and produces long-term detrimental effects on child health. The present study sought to evaluate the cytotoxic and genotoxic effects of chronic PCB exposure on fetal cells during pregnancy.

Plasma membrane and acrosome loss before ICSI is required for sheep embryonic development.

Purpose: This study aims to determine if the integrity of the sperm plasma membrane and acrosome vesicle could be limiting factors in sheep intracytoplasmic sperm injection (ICSI).

Methods: Prior to in vitro fertilization (IVF) or ICSI, the oocytes were subjected to in vitro maturation (IVM) for 24 h. First, to evaluate the need of artificial activation for ovine ICSI, 226 oocytes were injected with intact spermatozoa (IS), from which 125 were activated by incubation in ionomycin and 101 were cultured without activation.