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Introduction: Sperm cryopreservation is a valuable technique for storing valuable canine genetics. However, little is known concerning the fate of frozen sperm stored in a sperm bank. This study aimed to characterize dogs presented for sperm cryopreservation and describe the use and popularity of frozen sperm in the Netherlands and Belgium over recent years. Methods: Medical records from dogs presented for sperm cryopreservation between January 1, 2014 and December 31, 2022 at two different freezing centers were reviewed retrospectively. Imported frozen sperm was excluded due to lack of usage information. Each sperm cryopreservation was considered a single event, and data were collected separately for each cryopreserved sample. Results: A total of 3,090 ejaculates from 1,040 males of 157 different breeds were included and investigated using exploratory data analysis. The findings showed a steady rise in the popularity of sperm cryopreservation, with annual growth rates ranging from 8.4 to 41.9%. The majority of dogs (88.5%) were between 1 and 9 years old at the time of sperm cryopreservation, with nearly one-third aged 2–4 years. Most dogs were collected for sperm cryopreservation once (62.8%) or twice (21.6%). Sperm banks were used for both short- and long-term storage needs, and 6.83% of ejaculates were eventually discarded. The primary use of froze sperm was for international shipment, while 21.8% was used locally for artificial insemination. Depending on the year of cryopreservation, between 44.1 and 79.6% of frozen ejaculates remained unused or only partially used at the time of data collection. Discussion: The results of this study provides the first comprehensive analysis of the use and popularity of frozen sperm among dog breeders in Belgium and the Netherlands and suggest a change in breeding practices in recent years. The percentage of breeders resorting to sperm cryopreservation and the extent of frozen sperm use in current breeding strategies remain to be defined in future studies. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) contain microRNAs (miRNAs), which are important regulators of embryonic development. Nevertheless, little is known about the precise molecular processes controlling blastocyst development and quality. In a previous study, we identified bta-miR-665 as one of the miRNAs more abundantly present in extracellular vesicles of embryo-conditioned culture media of blastocysts compared to degenerate ones. Here, we investigated the effect and regulatory roles of bta-miR-665 in blastocyst development by supplementation of bta-miR-665 mimics or inhibitors to the culture media. Supplementation of bta-miR-665 mimics improved cleavage and blastocyst rate (P < 0.01), and blastocyst quality as indicated by increased inner cell mass rates and reduced apoptotic cell ratios (P < 0.01). Furthermore, supplementation of bta-miR-665 inhibitors had the opposite effect on these phenotypes. Low input transcriptome analysis and RT-qPCR revealed that bta-miR-665 acts on genes linked to microtubule formation and apoptosis/cell proliferation. These insights not only elucidate the important role of bta-miR-665 in embryo development, but also underscore its potential in improving reproductive efficiency in bovine embryo culture. [ABSTRACT FROM AUTHOR]

Introduction: The increasing prevalence of infertility in male dogs in clinical practice mirrors current trends seen in human medicine. Acquired infertility is notably more common in dogs compared to congenital causes, with conditions such as testicular degeneration leading to irreversible loss of fertility. Current diagnostic methods for testicular degeneration, such as histopathological and cytological examinations, rely on testicular biopsy or fine needle aspiration, making them less feasible for routine use. Anti-Müllerian hormone (AMH), produced by Sertoli cells, has emerged as a potential alternative biomarker for testicular health, which can be measured in serum. This study evaluates AMH as a potential marker for testicular degeneration, using cryptorchid dogs as models for impaired fertility and altered testicular histology. Methods: The relationship between serum AMH levels and AMH tissue expression with impaired spermatogenesis and altered histology was investigated. Serum AMH levels were determined in intact, cryptorchid, and castrated individuals using an immuno-enzymatic ELISA kit and compared between subgroups based on testicular location. Tissue AMH immuno-expression was differentially quantified in two regions of interest (ROIs), the interstitial space and the seminiferous tubule, in both descended and retained gonads. Furthermore, testicles were analyzed using histomorphometric analysis in seminiferous tubules, while spermatogenesis was evaluated using the Johnsen score. Results: Serum AMH levels were positively correlated with AMH expression assessed in both interstitial space (ρ = 0.494, p ≤ 0.01) and seminiferous tubules (ρ = 0.610, p ≤ 0.001). Conversely, serum AMH levels showed a negative correlation with the seminiferous tubule area (ρ = -0.435, p ≤ 0.05). Smaller seminiferous tubule areas were linked to increased AMH reactivity in both seminiferous tubules (ρ = -0.774, p ≤ 0.001) and interstitial space (ρ = -0.725, p ≤ 0.001). Additionally, lower Johnsen scores were associated with higher serum AMH levels (ρ = -0.537, p ≤ 0.01) and elevated AMH expression in both seminiferous tubules (ρ = -0.756, p ≤ 0.001) and interstitial space (ρ = -0.679, p ≤ 0.001). Discussion: Our results suggest that higher serum levels and tissue expression of AMH are linked to smaller seminiferous tubules and poorer Johnsen scores, reflecting degenerative changes and Sertoli cell dysfunction in retained testicles. Given the similarities in the mechanisms that increase AMH levels in both cryptorchid and non-cryptorchid testicles affected by testicular degeneration, this study recommends using AMH as a marker for diagnosing testicular degeneration in dogs. [ABSTRACT FROM AUTHOR]

Transfer RNA-derived small RNAs (tsRNAs) have been shown to be involved in early embryo development and repression of endogenous retroelements in embryos and stem cells. However, it is unknown whether tsRNAs also regulate embryo hatching. In this study, we mined the sequencing data of a previous experiment in which we demonstrated that the microRNA (miRNA) cargo of preimplantation embryonic extracellular vesicles (EVs) influences embryo development. We thus profiled the tsRNA cargo of EVs secreted by blastocysts and non-blastocysts. The majority of tsRNAs was identified as tRNA halves originating from the 5´ ends of tRNAs. Among the 148 differentially expressed tsRNAs, the 19 nt tRNA fragment (tRF) tDR-14:32-Glu-CTC-1 was found to be significantly up-regulated in EVs derived from non-blastocysts. RT-qPCR assays confirmed its significant up-regulation in non-blastocyst embryos and their conditioned medium compared to the blastocyst group (P < 0.05). Inhibition of tDR-14:32-Glu-CTC-1 by supplementing antagomirs to the conditioned medium improved embryo hatching (P < 0.05). Transcriptomic analysis of embryos treated with tDR-14:32-Glu-CTC-1 antagomirs further showed differential expression of genes that are associated with embryo hatching and implantation. In summary, tDR-14:32-Glu-CTC-1 is up-regulated in non-blastocyst embryos and their secretions, and inhibition of tDR-14:32-Glu-CTC-1 promotes embryo hatching, while influencing embryo implantation-related genes and pathways. These results indicate that embryonic EVs containing specific tRFs may regulate preimplantation embryo development. [ABSTRACT FROM AUTHOR]