Comparative analysis of milt quality in the cultured and wild stocks of endangered Caspian brown trout, Salmo trutta caspius

The sperm motility characteristics (percentage of motile spermatozoa and duration of motility) and sperm production (spermatocrit, milt volume and sperm concentration) were measured in order to compare the milt quality between cultured and wild stocks of Caspian brown trout, Salmo trutta caspius. Our results showed that cultured brooders produce more dense milt than wild individuals. In contrast, the milt volume, percentage and duration of spermatozoa motility were higher in wild brooders than in cultured individuals. The aim of the present study was to assess the effect of captivity condition on milt quality of cultured males of Caspian brown trout.Keywords: Sperm density, sperm motility, Caspian brown trou

Regulace subcelulárního vápníku během gametogeneze ryb

Changes in the characteristics of spermatogenic and oogenic cells during gametogenesis may reflect a corresponding alteration in aspects of components such as calcium, which plays prominent roles in regulating a broad range of physiological events in animal reproduction. Basic information regarding distribution of intracellular calcium in different germ cells may provide better understanding of processes of reproduction in fish. The monthly testicular development in the cultured breeding stock of sterlet, Acipenser ruthenus, using histological and serum sex steroid was studied. Results showed four distinct phases including resting, pre-spawning, spawning and post-spawning. Hormonal profiles of 11-ketotestosterone (11-KT) showed peak, which indicated a seasonal pattern of gonadal development. The 11-KT concentration increased considerably during the spermatogenesis (pre-spawning phase) and remained quite high throughout the pre-spermiation period. In the final phase of testicular development (spawning phase), the 11-KT markedly dropped. This study provides basic knowledge of the reproductive biology in male sterlet and a complete guide for gonadal development, heretofore lacking in previous studies of sturgeon gonadal development. The intracellular distribution of calcium during different developmental stages of spermatogenesis was studied in sterlet, A. ruthenus, using a combined oxalate-pyroantimonate technique. The distribution of calcium was described in spermatogonium, spermatocyte, spermatid, and spermatozoon stages. Although calcium is appeared in the form of deposits in limited areas of the early stages cells (spermatogonium and spermatocyte), it is present as an unbound form in larger areas of spermatids and spermatozoa especially nucleus, which probably reflects changes in its physiological function and homeostasis of calcium during male gamete production. Similar to sterlet sturgeon, ultrastructural distribution of calcium during different developmental stages of spermatogenesis was described in a model organism, zebrafish (Danio rerio), using a combined oxalate-pyroantimonate technique. The subcellular distribution of intracellular calcium was detected as deposits mainly in the cytoplasm and the nucleus of the spermatogonium and spermatocyte. Interestingly, large amount of calcium was transformed from isolated deposits into an unbound pool (electrondense mass) within the nucleus of the spermatid and the spermatozoon. The alteration of intracellular calcium at different stage of D. rerio spermatogenesis can be related to specific function of each germ cell types during male gamete development. Unbound calcium in the nucleus of mature spermatozoon can be used for condensation of chromatin and induction of calcium wave during egg activation and fertilization. Using a combined oxalate-pyroantimonate technique, the subcellular distribution of calcium deposits during stages of oogenesis in zebrafish was described. Calcium deposits were localized at different organells within the egg during oocyte development. At the first stage of oocyte development (primary growth), calcium deposits were localized in the cytoplasm, mitochondria, nucleus, and follicular cells. At the cortical-alveolus stage, calcium particles were transported from follicular cells into the cortical alveoli. In the main stage of oocyte development (vitellogenic stage), some cortical alveoli were compacted and transformed from flocculent electron-lucent to electron-dense objects with the progression of the stage. Calcium deposits were transformed from larger to smaller particles, coinciding with compaction of cortical alveoli. As a conclusion, results of this study provide new and convergent insight into information about the regulation and functional roles of calcium during fish gametogenesis which simply describe facilitate release of calcium from related organelles (nucleus and cortical alveoli) as main internal stores of calcium for calcium transport

In vitro antioxidant enzyme activity and sperm motility at different temperatures in sterlet Acipenser ruthenus and rainbow trout Oncorhynchus mykiss

Influence of in vitro temperature on sperm antioxidant enzyme activity, thiobarbituric acid-reactive substance (TBARS) content and motility parameters was evaluated in sterlet Acipenser ruthenus and rainbow trout Oncorhynchus mykiss. Sperm activation was conducted at 4, 14 and 24 degrees C in both species. Duration of motility was significantly longer at 4 degrees C than at 14 and 24 degrees C in both species. At 60 s post-activation, the velocity of sterlet spermatozoa was highest at 24 degrees C. This trend continued to 420 s post-activation. In rainbow trout, at 10 s post-activation, the highest velocity was observed at 14 degrees C. Significantly higher catalase activity was seen at 4 degrees C in both species. No significant difference in spermatozoon superoxide dismutase activity among temperatures was observed. In sterlet, TBARS content was significantly higher at 24 degrees C compared to other temperatures, but, in rainbow trout, it was highest at 4 degrees C. The results presume species-specific level of antioxidant enzyme activity and TBARS content at studied temperatures.</p

Quantification of egg proteome changes during fertilization in sterlet Acipenser ruthenus

Eggs of sterlet are discharged outside into ambient aquatic environment where egg activation and fertilization occur. Effects of different activation media including freshwater and clay suspension on protein abundances of egg were quantified in sterlet Acipenser ruthenus. In-gel digestion and high resolution mass spectrometry were used for label-free protein quantification in the eggs of five females. No significant (p > 0.05) difference was found between protein abundances in eggs activated with different media. However, results showed significant (p < 0.05, fold change ≥2) reduction in the abundances of nine proteins including six glycoproteins, enolase and heat shock protein in activated groups compared to freshly ovulated eggs as control. The fact that abundance of proteasome subunit alpha significantly reduced only in eggs which were activated by clay suspension suggests that activation medium can somehow intervene with protein regulation during fertilization. In conclusion, external fertilization in sturgeon egg is accompanied by huge release of proteins into the external environment that may participate in the construction of a transient microenvironment around egg for attraction and protection of spermatozoa to ensure ensuing fertilization. Data are available via ProteomeXchange with identifier PXD006232

Ultrastructural feature of spermatogenic cells and spermatozoon in cultured burbot Lota lota

Testis development and ultrastructure of spermatogenic cells and spermatozoa of burbot Lota Iota, a commercially important cold freshwater fish, were studied by light and transmission electron microscopy. Spermatogonia, spermatocytes, spermatids, and spermatozoa are distributed along the seminiferous tubules. Electron-dense bodies appear in germ cells from primary spermatogonia to secondary spermatocytes. We identified three distinct stages of spermatid cell differentiation based on chromatin condensation, development of the flagellum, formation of a nuclear fossa, and elimination of excess cytoplasm. Spermatozoa were anacrosomal and characterized by location of the centrioles outside the nuclear fossa and incomplete perpendicular arrangement of the centrioles. The sperm flagellum displayed an axoneme with nine doublets of peripheral microtubules and two central microtubules. These results provide valuable information for burbot taxonomy and may clarify the process of spermatogenesis for this species.</p

Effects of temperature on sperm motility of burbot Lota lota: spontaneous activation and calcium dependency

Several factors regulating activation of spermatozoon motility in Eurasian burbot, Lota lota, including osmolality, calcium (Ca2+) ions, and temperature were investigated. Spermatozoon motility in Eurasian burbot, Lota lota was assessed at 4 and 30 degrees C in seminal fluid, isotonic media (with and without Ca2+) and hypotonic media (with and without Ca2+). Spermatozoa were spontaneously activated in seminal fluid at 20 degrees C and the maximum motility was recorded at 30 degrees C, which is out of the spawning temperature range, indicating that no risk of activation occurs during routine semen handling in artificial insemination. Initiation of spermatozoon motility in L. lota is mediated by Ca2+ and sensitivity to Ca2+ is dependent on temperature.</p

Determination of annual reproductive cycle in male sterlet, Acipenser ruthenus using histology and ultrasound imaging

International audienceThe aim of this study was to evaluate seasonal testicular development in the cultured sterlet, Acipenser ruthenus. During annual sexual cycle of male sterlet, stages of gonad maturity were examined using histology and ultrasonography approaches. The histology identified males at different stages of maturity among fish sampled monthly. According to the seasonal changes in the testes, reproductive cycle was divided into four stages including resting, pre-spawning, spawning and post-spawning. The histology examination revealed considerable variation in testicular developmental stages. These changes were identified based on persistent spermatogenesis and asynchronous gonad development in testes, showing regulation of annual gonadal cycle is influenced by season. Also, the results obtained using ultrasound suggested that reproductive stages can be identified based on morphology and tissue echogenicity. At each phase of testicular development, gonadosomatic index (GSI) and number of spermatogenic cysts were variable. The present study focused on determination of annual reproductive development in cultured male sterlet which clearly identifies reproductive stage in each season as valuable guide for future researches on reproductive biology of sterlet. This study presents basic knowledge about reproductive biology in sterlet contributing to optimal broodstocks management that allows comparison of reproductive development among sturgeon species

Efficient generation of zebrafish maternal-zygotic mutants through transplantation of ectopically induced and Cas9/gRNA targeted primordial germ cells

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology has been widely utilized for knocking out genes involved in various biological processes in zebrafish. Despite this technology is efficient for generating different mutations, one of the main drawbacks is low survival rate during embryogenesis when knocking out some embryonic lethal genes. To overcome this problem, we developed a novel strategy using a combination of CRISPR/Cas9 mediated gene knockout with primordial germ cell (PGC) transplantation (PGCT) to facilitate and speed up the process of zebrafish mutant generation, particularly for embryonic lethal genes. Firstly, we optimized the procedure for CRISPR/Cas9 targeted PGCT by increasing the efficiencies of genome mutation in PGCs and induction of PGC fates in donor embryos for PGCT. Secondly, the optimized CRISPR/Cas9 targeted PGCT was utilized for generation of maternal-zygotic (MZ) mutants of tcf7l1a (gene essential for head development), pou5f3 (gene essential for zygotic genome activation) and chd (gene essential for dorsal development) at F-1 generation with relatively high efficiency. Finally, we revealed some novel phenotypes in MZ mutants of tcf7l1a and chd, as MZtcf7l1a showed elevated neural crest development while MZchd had much severer ventralization than its zygotic counterparts. Therefore, this study presents an efficient and powerful method for generating MZ mutants of embryonic lethal genes in zebrafish. It is also feasible to speed up the genome editing in commercial fishes by utilizing a similar approach by surrogate production of CRISPR/Cas9 targeted germ cells. Copyright (C) 2020, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved