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Sperm metabolism consists of a sophisticated network of biochemical reactions and varies between species, resulting in different metabolic strategies for ATP production to maintain sperm functionality. ATP can be produced through glycolysis or in the mitochondria by oxidative phosphorylation (OXPHOS). Since OXPHOS is the predominant metabolic pathway in horses spermatozoa, various assessments of mitochondrial activity are used to evaluate fertility, utilizing techniques such as fluorescent probes analysed via microscopy or flow cytometry, and polarographic electrode assays to measure current flow in response to an applied voltage. Though, these methods are limited by low throughput, as they assess mitochondrial activity at a single time point under a specific treatment condition. This study explores, for the first time, the application of the Agilent Seahorse XFp Technology to evaluate metabolism in stallion spermatozoa. This method enables real-time measurement of cellular metabolism across multiple samples or experimental conditions simultaneously. Ejaculates from eight different stallions were collected, and pools were prepared from three of them. Sperm viability and mitochondrial activity were evaluated by fluorescence microscopy, sperm motility by a computer-assisted sperm analysis system, and sperm metabolism was analysed via the Seahorse XFp analyser. Results confirmed a preference for OXPHOS over glycolysis in ATP production in stallion sperm, with mitochondria contributing significantly to total ATP generation. The Seahorse XFp Technology proved effective in evaluating equine sperm bioenergetics, offering insights into metabolic pathways critical for sperm function. In conclusion, this technology grants a new method for high-throughput analysis of sperm metabolism and quality, which could be applied to future reproductive studies in male equine fertility., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Artificial reproduction is a bottleneck to produce stocking material for many species of freshwater fish. One of these species is the asp, Leuciscus aspius. Research in the field of artificial reproduction of this species is very scarce and often incomplete. There are no breeding protocols specifying optimal environmental conditions and hormonal stimulation for many species of rheophilic cyprinids, including asp. Since the number of natural asp populations is constantly decreasing, it is important to support natural stocks by restocking with high quality stocking material. For this reason, optimized protocols are needed to breed this species under controlled conditions to produce stocking material with high biodiversity and good health. Such an approach will make it possible to maintain the population of natural asp at a constant level. The aim of this study was to develop the protocol of asp artificial reproduction using optimized thermal conditions and appropriate hormonal stimulation. In experiment I, the influence of constant temperature (10.0, 12.0 and 14.0 °C) on the effectiveness of artificial reproduction of asp. In experiment II, the effectiveness of asp reproduction was checked after the application of spawning agents: Ovopel, Ovaprim or a combination of these two agents The obtained results indicate that for the final maturation of oocytes (FOM) and artificial reproduction of asp in controlled conditions, water temperatures of 10-12 °C are the most useful. Under these thermal conditions, the highest percentages of female's ovulation and embryo survival, as well as the percentage of hatching, were obtained. Hormone injections are necessary to perform final oocyte maturation (FOM) in female asp in captivity. All spawning agents used were especially useful for artificial reproduction of asp, however, the best values of the studied indices, such as ovulation rate and embryo survival, were obtained after the application of Ovaprim or the combination of Ovopel and Ovaprim in water temperature at a range of 10-12 °C. It was found that the pH of ovarian fluid may be a preliminary indicator of the biological quality of eggs in asps. The optimal pH value is 8.0-8.4. At pH below 7.4, no viable embryos were observed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This paper describes a study in which different types of the reproduction of wild vimba bream (Vimba vimba) under controlled conditions were compared. Reproduction was performed by using three methods: spontaneous (natural spawning- NS groups): without hormones but with nests, semi-artificial (S groups) with hormones and nests, and artificial (A groups) with hormones and manual stripping of gametes. In groups S and A, different types of hormonal stimulation were tested: common carp pituitary homogenate (CPH), human chorionic gonadotropin (hCG), and commercial pellets containing mammalian GnRHa and dopamine antagonist-metoclopramide (Ovopel). The number of spawning fish, i.e., spermiating males and ovulating females, were compared depending on the type of breeding and the stimulant used. The lowest number of finally matured fish: spermiating males (67 %) and ovulating females (0 %) were noted in the spontaneously reproduced group. The best method of reproducing wild vimba bream under controlled conditions was the semi-artificial approach with hormonal stimulation and spawning nests, where ovulation rates in hormonally treated groups were between 43 % and 71 %, vs 26-57 % in artificial spawning groups. No ovulation observe in control groups in S and A types of reproduction. The highest embryo survival to the eyed-egg-stage was noted in group A-hCG (average 88.2). A higher value of spawning effectiveness (Se) with the use of the same hormones was obtained for groups S (0.374-0.574) than for groups A (0.252-0.493). In both cases, the highest Se was recorded for Ovopel stimulated fish and the lowest in hCG stimulated groups., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Seasonal reproduction of domesticated pikeperch has been the most critical spawning batch in several European countries. The present study aimed to monitor oocyte growth between mid-November and seasonal spawning to evaluate if oocyte growth trends may predict egg quality. Nineteen sexually mature females were monitored for oocyte sizes every two months. In mid-March, fish were transported to the indoor facility and artificially reproduced. Sixteen females ovulated and egg quality parameters were assessed and further related to oocyte sizes measured in November, January, and March, as well as to size increments between samplings. Based on the oocyte diameters, fish were assigned to two size groups, and the egg quality was compared. Oocyte growth was greater between the first two samplings compared to the consecutive period (150.1 ± 16.5 µm vs 24.7 ± 20.4 µm). Diameters assessed in November and March positively correlated with oil globule fragmentation, while diameters assessed in November and January positively correlated with larval malformation. Although in January larger oocytes showed better embryo survival than smaller ones (60.6 ± 9.5% vs 37.8 ± 23.2%,) they were characterized with greater percent of malformed larvae (25.0 ± 22.0% vs 5.4 ± 3.9%). It appears that fish with bigger oocytes in pre-season have an affinity toward increased embryo survival, however, they seem to have an issue with oil globule fragmentation and larvae malformation in case of seasonal spawning. Therefore, the selection of breeders for either pre-seasonal or seasonal reproduction batch according to the November oocyte size is recommended., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The aim of this study was to evaluate the efficiency of different hormonal agents and their combinations for spawning induction of females Caspian roach, Rutilus caspicus. Five groups of fish were injected intraperitoneally as follows: two injections of Ovaprim (T1), two injections of Ovopel (T2), a priming dose of Ovopel with a resolving dose of Ovaprim (T3), a priming dose of Ovaprim with a resolving dose of Ovopel (T4), and two injections of sterile 0.9% NaCl solution as a control group. The results showed that no fish ovulated in the control group. In hormone-treated groups, no differences were observed in ovulation success (P > 0.05). The shortest latency time and highest working and relative fecundities were observed in fish stimulated with two injections of Ovaprim (T1 group). The mean values of fertilization success in treated fish were in the range of 87.9-93.1% and the highest values were observed in T3 and T1 groups (P < 0.05). The best results in terms of ovulation index (92.1%), hatching rate (93.7%), and survival up to the eyed egg stage (89.4%) were achieved after administering two injections of Ovaprim (T1 group). There were no significant differences in the incubation time and embryos survival rates up to absorbing the yolk sac among different groups (P > 0.05). Overall, the best results of controlled reproduction of Caspian roach were obtained after using two injections of Ovaprim. Consequently, the Ovaprim is more suitable for restocking and aquaculture purposes of endangered populations of Caspian roach., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Use of carp pituitary extract (CPE) as a hormone inducer for Leporinus obtusidens is cost-prohibitive; moreover, CPE contains unknown concentrations of gonadotropins. We evaluated the efficacy of gonadorelin as a substitute by analyzing its effect on sperm characteristics of L. obtusidens and cost-effectiveness (20, 40, 60, 80, and 100μgkg -1 ). Untreated and CPE-treated (4mgkg -1 ) fish were used as negative and positive controls, respectively. Following a single intramuscular dose of hormones, semen was collected at 200°-h. At doses from 60μgkg -1 , all fish produced higher milt volume (P<0.05) than the negative control (0.2-0.8mLkg -1 ), equal (P>0.05) to the positive control (3.52±0.97mLkg -1 ). Gonadorelin did not affect sperm concentration (P>0.05). The percentage of morphologically normal spermatozoa was >64% in all treatments. The number of spermatozoa with intact membrane (70%-85%) was equal (P>0.05) to that in the controls. Relative to the negative control, gonadorelin did not affect the motility rate (P>0.05). Curvilinear velocity was equal (P>0.05) to that in both controls. The sperm swimming path was linear (0.9) 10s after activation and was not (P>0.05) dose-dependent. Considering the response rate of gonadorelin-induced fish and the similarity of semen quality compared to controls, the lowest effective dose to produce a higher volume of semen in L. obtusidens is 60μgkg -1 , and the cost to induce 1kg of L. obtusidens is 14.0% lower than that with CPE., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Standardization of fertilization protocols for sterlet Acipenser ruthenus is crucial for improving reproductive techniques and for conservation purposes. Our objectives were to determine the number of sperm (tested 430,000:1, 43,000:1, 4300:1, 430:1 sperm to egg) required to fertilize eggs and explore how pre-incubation of eggs in freshwater for 0min, 0.5min, 1min, and 10min interacts with different sperm ratios. Fertilization success ranged from 29.7% at 430:1 to 84.2% at 430,000:1. Pre-incubation time had no effect on fertilization success at 430,000:1 and 43,000:1 sperm to egg ratios, while it was significant at the 4300:1 and 430:1 ratios. The use of adequate experimental suboptimal sperm to egg ratio revealed a positive effect of pre-incubation time, such that at the 430:1 ratio, 0.5min pre-incubation increased the fertilization rate than 10min. At 0min pre-incubation the proportion of fertilized eggs increased at the 430,000:1 ratio, while at 1min fertilization increased at the 4300:1 ratio. At the 10min pre-incubation time, fertilization increased at the 43,000:1 ratio. Moreover, at the 0.5min pre-incubation time, the 43,000:1 ratio increased the fertilization rate than the 430:1 ratio. Generally, for 430:1 ratio, the fertilization rate is lower than in control. Transmission electron microscopy showed that pre-incubation of eggs in water for <10min does not trigger a cortical reaction or the formation of a perivitelline space. Results suggest that with a low sperm to egg ratio 0.5 to 1min pre-incubation of eggs in freshwater prior to fertilization can enhance fertilization rate of sterlet., (Copyright © 2015 Elsevier B.V. All rights reserved.)