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Bulls used in cattle production are often overfed to induce rapid growth, early puberty and increase sale price. While the negative consequences of undernutrition on bull sperm quality are known, it is unclear how a high gain diet influences embryo development. We hypothesized that semen collected from bulls fed a high gain diet would have a reduced capacity to produce blastocysts following in vitro fertilization. Eight mature bulls were stratified by body weight and fed the same diet for 67 d at either a maintenance level (0.5% body weight per day; n = 4) or a high gain rate (1.25% body weight per day; n = 4). Semen was collected by electroejaculation at the end of the feeding regimen and subjected to sperm analysis, frozen, and used for in vitro fertilization. The high gain diet increased body weight, average daily gain, and subcutaneous fat thickness compared to the maintenance diet. Sperm of high gain bulls tended to have increased early necrosis and had increased post-thaw acrosome damage compared with maintenance bulls, but diet did not affect sperm motility or morphology. Semen of high gain bulls reduced the percentage of cleaved oocytes that developed to blastocyst stage embryos. Paternal diet had no effect on the number of total or CDX2 positive cells of blastocysts, or blastocysts gene expression for markers associated with developmental capacity. Feeding bulls a high gain diet did not affect sperm morphology or motility, but increased adiposity and reduced the ability of sperm to generate blastocyst stage embryos.

Sperm transport through the female reproductive tract requires energy for motility and fertilization. Sperm kinematic assessment is conducted as an industry standard to estimate semen quality prior to bovine insemination. However, individual samples with similar post-thaw motility result in different pregnancy outcomes, suggesting that differences in bioenergetics may be important for sperm function. Thus, characterization of bioenergetic and kinematic parameters of sperm over time may reveal novel metabolic requirements for sperm function. Post-thawed sperm from five samples of individual (A, B, C) and pooled bulls (AB, AC) were assessed at 0 and 24 hr after thawing. Sperm were evaluated for kinematics via Computer-Assisted Sperm Analyses and bioenergetic profiles using a Seahorse Analyzer for basal respiration [BR], mitochondrial stress test [MST] and energy map [EM]. Motility was nearly identical among samples after thawing and no differences in bioenergetics were detected. However, after 24 hr of sperm storage, pooled sperm samples (AC) presented with higher BR and proton leakage compared to other samples. Sperm kinematic variability among samples was higher after 24 hr, suggesting difference in sperm quality may manifest over time. Despite a reduction in motility and mitochondrial membrane potential, BR was higher at 24 hr compared to 0 hr for nearly all samples. A metabolic divergence between samples was detected by EM, indicating a shift in bioenergetic profiles over time that was undetected after thawing. These new bioenergetic profiles elucidate a novel dynamic plasticity of sperm metabolism over time while suggesting an influence of heterospermic interactions for further investigation.

Containers used to cryopreserve sperm are made with non-biodegradable plastic compounds, having a high monetary and environmental cost. Therefore, the development of biodegradable alternative containers for cell cryopreservation is necessary. Thus, this study aimed to evaluate the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as low-cost and biodegradable alternative containers for sperm cryopreservation. Sperm from twelve South American silver catfish Rhamdia quelen were individually cryopreserved in plastic straws 0.25 mL (as control), hard-gelatin, and hard-HPMC capsules. The quality of post-thaw sperm cryopreserved in the different containers was checked by measuring spermatozoa membrane integrity, kinetic parameters, mitochondrial activity, fertilization, hatching, and normal larvae rates. The samples cryopreserved in straws showed a higher percentage of membrane integrity (68%) than those frozen in hard-gelatin (40%) and hard-HPMC capsules (40%). However, we did not observe differences between the samples stored in straws and hard capsules for the rest of tested sperm parameters. Thus, based on the high sperm fertility capability, both capsules were efficient as cryopreservation containers for maintaining sperm functionality.

Primordial germ cells (PGCs) development is a subtle and complex regulatory process. Fance is an important substrate molecule necessary for the activation of the Fanconi anemia (FA) pathway, and its homozygous mutant causes massive oocyte loss as early as embryonic day 13.5 (E13.5). Here, we present histological and RNA-seq analysis of Fance deficient PGCs to explore the possible mechanisms responsible for its progressive depletion of germ cells. In Fance^-/- embryos, the reduction of PGCs was already evident at E9.5 and the progressive loss of PGCs led to the PGCs being almost exhausted at E12.5. An increase of apoptotic cells was detected among Fance^-/- PGCs, which may intuitively explain their reduced number in embryos. Moreover, abnormal cell proliferation and accumulating DNA damage were detected in E12.5 Fance^-/- PGCs. We identified 3026 differentially expressed genes in E12.5 Fance^-/- PGCs compared to Fance^+/+ . KEGG pathway analysis revealed that the up-regulated genes were highly associated with ‘lysosome’, various metabolism pathways, whereas the down-regulated genes were mainly enriched in ‘cell cycle’, ‘oocyte meiosis’, ‘ribosome’, and various DNA repair pathways. In addition, multiple genes of various cell death pathways were found to be differentially expressed in E12.5 Fance^-/- PGCs, indicating that PGCs death in Fance^-/- embryos might diverge from canonical apoptosis. These findings indicate that Fance is essential for PGCs survival and the potential mechanisms involve cell cycle regulation, DNA damage repair, cell death prevention, and by regulating lysosome and ribosome function. Our results provide an important reference for further studies.