Effect of Sperm Cryopreservation on miRNA Expression and Early Embryonic Development

Although sperm preservation is a common means of personal fertility preservation, its effects on embryonic development potential need further investigation. The purpose of this study was to identify key microRNA (miRNA) in cryopreserved sperm and determine the changes of these miRNAs and their target genes during embryonic development using cryopreserved sperm. Moreover, the embryonic development potential of cryopreserved sperm was estimated in assisted reproductive technology (ART), where key miRNAs and target genes were validated in sperm and subsequent embryos. Clinical data of embryonic development from cryopreserved sperm indicated a significant decrease in fertilization rate in both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cases, as well as a reduction in blastocyst formation rate in ICSI cases. Meanwhile there was a significant increase in blocked embryo ratio of Day1, Day2, and Day3.5 embryos when frozen-thawed mouse sperm was used, compared with fresh mouse sperm, suggesting a potential negative effect of sperm cryopreservation on embryonic development. From frozen-thawed and fresh sperm in humans and mice, respectively, 21 and 95 differentially expressed miRNAs (DEmiRs) were detected. miR-148b-3p were downregulated in both human and mouse frozen-thawed sperm and were also decreased in embryos after fertilization using cryopreserved sperm. Target genes of miR-148b-3p, Pten, was identified in mouse embryos using quantitative real-time PCR (qRT-PCR) and Western blot (WB). In addition, common characters of cryopreservation of mouse oocytes compared with sperm were also detected; downregulation of miR-148b-3p was also confirmed in cryopreserved oocytes. In summary, our study suggested that cryopreservation of sperm could change the expression of miRNAs, especially the miR-148b-3p across humans and mice, and may further affect fertilization and embryo development by increasing the expression of Pten. Moreover, downregulation of miR-148b-3p induced by cryopreservation was conserved in mouse gametes.

L-Carnitine Improves Cytoprotection during Cryopreservation: A case study on Nili-Ravi Buffalo Sperm

The current study was aimed to evaluate the antioxidative effect of L-Carnitine at post thawing following cryopreservation of Nili-Ravi buffalo sperm. For this purpose, semen from three buffalo bulls were collected for 3 weeks using an artificial vagina (N=18; replicates). The qualified ejaculates were diluted employing tris-citric acid extender i.e., control did not receive any L-Carnitine and experimental groups having 0.5, 1.0, and 1.5 ng/mL of L-carnitine at 37 C with approximately 50 x 106 sperm/mL. The semen was cooled at 4 C and then equilibrated (4 hours), filled in straws (0.5 mL) at4 C, placed on LN2 vapours for 10 min, and kept into an LN2 container. The thawed semen was evaluated for post-thaw quality. The integrity of the sperm plasma membrane and motility (P?0.05) was highest in the extenders having 1.0 ng/mL of L-carnitine as compared to the control(received no L-Carnitine). However, sperm chromatin integrity and viability(live sperm with intact acrosome) remained similar. It was concluded that supplementing 1.0 ng/mL L-Carnitine of extender can improve the post-thaw quality of cryopreserved sperm. Based on the results of the current experiments it is recommended to include L-carnitine extender to improve post-thaw quality of buffalo sperm in terms of its motility and integrity ofits plasma membrane. Keywords: Buffalo, Sperm, Cryopreservation, Extender, L-Carnitine, Artificial insemination.

Assisted gene flow using cryopreserved sperm in critically endangered coral

Assisted gene flow (AGF) is a conservation intervention to accelerate species adaptation to climate change by importing genetic diversity into at-risk populations. Corals exemplify both the need for AGF and its technical challenges; corals have declined in abundance, suffered pervasive reproductive failures, and struggled to adapt to climate change, yet mature corals cannot be easily moved for breeding, and coral gametes lose viability within hours. Here, we report the successful demonstration of AGF in corals using cryopreserved sperm that was frozen for 2 to 10 y. We fertilized Acropora palmata eggs from the western Caribbean (Curaçao) with cryopreserved sperm from genetically distinct populations in the eastern and central Caribbean (Florida and Puerto Rico, respectively). We then confirmed interpopulation parentage in the Curaçao–Florida offspring using 19,696 single-nucleotide polymorphism markers. Thus, we provide evidence of reproductive compatibility of a Caribbean coral across a recognized barrier to gene flow. The 6-mo survival of AGF offspring was 42%, the highest ever achieved in this species, yielding the largest wildlife population ever raised from cryopreserved material. By breeding a critically endangered coral across its range without moving adults, we show that AGF using cryopreservation is a viable conservation tool to increase genetic diversity in threatened marine populations.

From the Semen Collection Method to the Hatchlings: The Use of Cryopreserved Sperm from Pheasants Fed an Antioxidant-Enriched Diet

A widely used approach to preserving genetic diversity in birds involves the cryopreservation of semen. In this process, cells are subjected to physical and chemical stresses, but not all cell species respond equally. Many studies have been published on the freezing–thawing of sperm cells from a wide variety of domestic and wild species, on issues ranging from the sperm quality to different protocols, fertilisation success rates, etc. Nevertheless, very little information is available on the common pheasant. To fill this gap, the aim of this study was to describe the pheasant semen collection method, evaluate some qualitative parameters of sperm from males fed an antioxidant-enriched diet, and to test the in vivo fertilising capacity of the cryo-preserved semen. The freezing protocol employed involved pellets thawed by the hotplate method. Dimethylacetamide was used as a cryoprotectant at a final concentration of 6%. A total of six AIs were performed at 3-4-day intervals on a total of 40 females with doses of 35 × 106 of normal live thawed sperm. Males receiving the enriched diet produce more abundant and concentrated ejaculates. Freeze–thawed sperm lost 85% of their initial mobility, and diet influenced neither sperm mobility nor viability. The enriched diet did improve the number of normal freeze–thawed cells and was associated with a lower sperm fracture incidence. Regardless of the dietary group, frozen–thawed sperm resulted in a fertility rate of 30%, with 8-9 chicks hatching for every 100 eggs incubated.

A simple and economic protocol for efficient in vitro fertilization using cryopreserved mouse sperm v2

The advent of genome editing tools like CRISPR/Cas has substantially increased the number of genetically engineered mouse models in recent years. In support of refinement and reduction, sperm cryopreservation is advantageous compared to embryo freezing for archiving and distribution of such mouse models. The in vitro fertilization using cryopreserved sperm from the most widely used C57BL/6 strain has become highly efficient in recent years due to several improvements of the procedure. However, purchase of the necessary media for routine application of the current protocol poses a constant burden on budgetary constraints. In-house media preparation, instead, is complex and requires quality control of each batch. Here, we describe a cost-effective and easily adaptable approach for in vitro fertilization using cryopreserved C57BL/6 sperm. This is mainly achieved by modification of an affordable commercial fertilization medium and a step-by-step description of all other necessary reagents. This protocol is compatible with frozen sperm from all major repositories and the IVF can easily be adapted to accommodate freshly harvested sperm.