Developmental competence of eggs produced by rainbow trout Doubled Haploids (DHs) and generation of the clonal lines

Poor quality eggs produced by the fully homozygous doubled haploids (DHs) may impair generation of clonal lines in fish species. In the present research, gynogenetic development of rainbow trout (Oncorhynchus mykiss) was induced in eggs originated from the DH females. Eggs were activated with the UV‐irradiated grayling (Thymallus thymallus) spermatozoa and subjected to the high hydrostatic pressure (HHP) shock to provide diploid clonal individuals. Only two of four DH females produced eggs that were successfully activated by the irradiated spermatozoa and subsequently developed into the gynogenetic embryos. Survival rates of rainbow trout from the clonal lines equalled 21.5% and 19.8% during embryogenesis and decreased after hatching to 18.6% and 14.9%, respectively. Some of the dead rainbow trout clones collected between hatching and swim‐up stage were emaciated and exhibited spinal deformities including scoliosis. Provided results confirmed limited developmental competences of eggs produced by rainbow trout DH females. Clonal rainbow trout developing in such eggs exhibited reduced survival and increased frequency of the body abnormalities.     

Induction of Gynogenetic and Androgenetic Haploid and Doubled Haploid Development in the Brown Trout (Salmo trutta Linnaeus 1758)

Gynogenetic and androgenetic brown trout (Salmo trutta Linnaeus 1758) haploids (Hs) and doubled haploids (DHs) were produced in the present research. Haploid development was induced by radiation‐induced genetic inactivation of spermatozoa (gynogenesis) or eggs (androgenesis) before insemination. To provide DHs, gynogenetic and androgenetic haploid zygotes were subjected to the high pressure shock to suppress the first mitotic cleavage. Among haploids, gynogenetic embryos were showing lower mortality when compared to the androgenetic embryos; however, most of them die before the first feeding stage. Gynogenetic doubled haploids provided in the course of the brown trout eggs activation performed by homologous and heterologous sperm (rainbow trout) were developing equally showing hatching rates of 14.76 ± 2.4% and 16.14 ± 2.90% and the survival rates at the first feeding stage of 10.48 ± 3.48% and 12.78 ± 2.18%, respectively. Significantly, lower survival rate was observed among androgenetic progenies from the diploid groups with only few specimens that survived to the first feeding stage. Cytogenetic survey showed that among embryos from the diploid variants of the research, only gynogenetic individuals possessed doubled sets of chromosomes. Thus, it is reasonable to assume that radiation employed for the genetic inactivation of the brown trout eggs misaligned mechanism responsible for the cell divisions and might have delayed or even arrested the first mitotic cleavage in the androgenetic brown trout zygotes. Moreover, protocol for the radiation‐induced inactivation of the paternal and maternal genome should be adjusted as some of the cytogenetically surveyed gynogenetic and androgenetic embryos exhibited fragments of the irradiated chromosomes.     

Comparison of Protein Fractions in Seminal Plasma from Multiple Sperm Collections in Sterlet (Acipenser ruthenus)

Seminal plasma of sterlet Acipenser ruthenus was evaluated using comparative proteomics to characterize its protein fractions and to determine any influence of multiple sperm collections on these proteins. An experimental group of fish was used, in which sperm was collected three times at 5 h intervals. Protein fractions of seminal plasma were determined by SDS‐gel electrophoresis (SDS‐PAGE) and two‐dimensional electrophoresis high‐resolution gels (2D). At all stripping times, five protein bands with molecular weights of 93, 53, 48, 33 and 28 kDa were identified using SDS‐PAGE. No significant differences (p > 0.05) in relative mass of protein bands among collections were observed. At the third collection, 20 protein spots were detected from the two‐dimensional gels, compared to 17 found at the first and second collections. Ten protein spots, from the third stripping, were analysed. Screening of these spots by mass spectrometric analysis showed positive results for spot 10. Direct comparison across public databases revealed sequence similarity with two hypothetical proteins, MCAG_00854 and IscW_ISCW011489. Differences in the seminal plasma protein fractions were found at the third stripping compared to the first two. It is hypothesized that these extra proteins after the third collection could be involved in some step of intracellular mechanism which is responsible for regulating of spermatozoa motility. However, protein identification revealed no significant distinction for any protein spot and protein sequences available in public databases. These results highlighted the need for a complete genome sequences for sturgeons.     

Absence of Sperm Factors as in the Parthenogenesis Does Not Interfere on Bovine Embryo Sensitiveness to Heat Shock at Pre‐Implantation Stage

Oocyte has been considered the major contributor for embryo thermo‐tolerance. However, it was shown that sperm factors can be transferred to the oocyte during fertilization, raising the question of whether the absence of such factors could interfere on embryo thermo‐tolerance. In this study, we used parthenogenesis to generate bovine embryos without spermatozoa in order to test whether the absence of sperm factors could influence their thermo‐sensitiveness at early stages. In vitro fertilized (IVF) and parthenogenetic (PA) embryos at 44 h post‐insemination/chemical activation were exposed to 38.5°C (control) or 41°C (heat shock) for 12 h and then developed for 48 h and up to blastocyst stage. Apoptosis index and expression of PRDX1, GLUT1, GLUT5 and IGF1r genes in blastocysts derived from heat‐shocked embryos were also evaluated. The heat shock decreased the blastocyst rate at day seven (p < 0.05) for IVF embryos and at day eight (p < 0.01) for both IVF and PA embryos. Total cell number was not affected by heat shock in IVF and PA blastocysts, but there was an increased proportion (p < 0.05) of apoptotic cells in heat‐shocked embryos when compared to controls. There was no interaction (p > 0.05) between method of activation (IVF and PA) and temperature (38.5°C or 41.5°C) for all developmental parameters evaluated. Expression of GLUT1 gene was downregulated (p < 0.05) by heat shock in both IVF and PA blastocyst whereas expression of GLUT5 and IGF1r genes was downregulated (p < 0.05) by heat shock in PA blastocysts. Those data show that the heat shock affects negatively the embryo development towards blastocysts stage, increases the apoptotic index and disturbed the expression of some genes in both IVF and PA embryos, indicating that the presence or absence of sperm factors does not influence the sensitivity of the bovine embryo to heat shock.     

Spermatozoa Concentration,Seminal Plasma Composition and Their Physiological Relationship in the Endangered Stellate Sturgeon (Acipenser stellatus) and Russian Sturgeon (Acipenser gueldenstaedtii )

Comparative studies of ionic composition, osmolality, protein concentration and pH of seminal plasma along with spermatozoa concentrations were carried out in stellate sturgeon, Acipenser stellatus, and Russian sturgeon, Acipenser gueldenstaedtii. Analysis of A. gueldenstaedtii sperm showed significantly higher concentrations of Na⁺ (34.58 ± 4.61 mm ), Ca²⁺ (0.35 ± 0.12 mm ), Mg²⁺ (0.70 ± 0.25 mm ), Cl^? (13.50 ± 4.04 mm ) and proteins (0.60 ± 0.29 mg/ml) in the seminal plasma than did seminal plasma of A. stellatus: Na⁺ (20.08 ± 10.75 mm ), Ca²⁺ (0.28 ± 0.06 mm ), Mg²⁺ (0.29 ± 0.05 mm ), Cl^? (7.50 ± 3.00 mm ) and 0.30 ± 0.11 mg/ml proteins. Significantly higher concentration of K⁺ (5.42 ± 1.06 mm ) was observed in A. stellatus compared to A. gueldenstaedtii K⁺ (2.29 ± 0.50 mm ). Concentration of Na⁺ was positively correlated with osmolality (r = 0.819), levels of Cl^? (r = 0.922) and Mg²⁺ (r = 0.727) and pH (r = 0.848). The concentration of Mg²⁺ was positively correlated with protein concentration (r = 0.774), Na⁺ (r = 0.727), Cl^? (r = 0.872) and Ca²⁺ (r = 0.801). A positive relationship was also found between concentration of K⁺ and spermatozoa concentration (r = 0.709). Results revealed strong inter‐species differences in several parameters. The data should be useful for artificial fertilization and for cryopreservation of sturgeon sperm.     

Retracted: Effects of multiple collections on spermatozoa quality of Persian sturgeon,Acipenser persicus: motility,density and seminal plasma composition

In this study, we investigated the effects of multiple collections of sperm on the endangered Persian sturgeon, Acipenser persicus, in terms of a number of sperm functional parameters (percentage of motile spermatozoa, total time period of motility and sperm concentration) as well as on the ionic composition, protein concentration and osmolality of seminal plasma. Semen samples were collected from 12 induced male fish in three experimental groups that had been injected intramuscularly with LHRH‐A₂, at dosages of 5 μg/kg body weight, at a number of time regimes: at 12 h, 17 h and 24 h after spawning induction (1); at 24, 29 and 34 h after spawning induction (2); and at 36, 41 and 46 h after spawning induction (3). The percentage of motile spermatozoa and the period of sperm motility decreased significantly (p < 0.05) after the second and third collections. The concentration of spermatozoa decreased after the third collection, but this decline was not significant. No significant effect of multiple collections on protein concentration and ionic content (with exception of the Cl^? ion) of seminal plasma was observed. In all experimental groups, a moderate impact of sequential collection on the osmolality (p < 0.05) of seminal plasma was observed. This study provides new data on the effects of multiple collections on spermatological characteristics in the Persian sturgeon. Our results confirm that sequential stripping after the third collections has a negative effect on a number of functional parameters associated with sperm.     

Contrasting effects of heat shock during in vitro maturation on development of in vitro‐fertilized and parthenogenetic bovine embryos

This study investigated the influence of heat shock during in vitro maturation on embryo development following in vitro fertilization (IVF) or parthenogenesis (Part). Immature bovine cumulus–oocyte complexes were exposed to heat shock (41.0°C) during the first 12 hr of in vitro maturation (IVM), followed by 12 hr at 38.5°C. Control group consisted of in vitro maturation for 24 hr at 38.5°C. Oocytes were in vitro‐fertilized or activated with ionomycin and cultured in vitro for 192 hr post‐in vitro insemination or parthenogenetic activation (hpia). There was an interaction (p < .01) between temperature of IVM and method of oocyte activation (IVF or Part) for cleavage at 48 hpia. Heat shock had a negative impact (p < .01) on cleavage of IVF embryos, whereas no (p > .05) effect was found in the Part embryos. Embryo development towards blastocyst stage at 168 and 192 hpia decreased in both IVF and Part embryos derived from heat‐shocked oocytes. Heat shock increased (p < .05) the apoptotic index in Part blastocysts, but no effect (p > .05) was found in IVF counterparts. Heat shock also down‐regulated the expression of AQP3 (p < .01) and up‐regulated the expression of HSP70.1 (p < .01) in Part blastocysts, whereas it down‐regulated the expression of ATP1A1 (p < .05) in IVF blastocysts. In conclusion, the effects of heat shock during IVM on early embryo cleavage and blastocyst apoptosis are influenced by the method of oocyte activation and expression of some genes can be disturbed in embryos derived from heat‐shocked oocytes.     

Different thermotolerances in in vitro‐produced embryos derived from different maternal and paternal genetic backgrounds

The present study evaluated the effects of genetic backgrounds on the developmental competence and thermotolerance of bovine in vitro‐produced (IVP) embryos. First, Holstein (Hol) and Japanese Black (JB) oocytes were fertilized with sperm from Hol, JB and a thermotolerant breed (Brahman), and in vitro development was evaluated when the embryos were exposed to heat shock on Day 2 (Day 0 = day of fertilization). Sperm genetic backgrounds affected the developmental competence in controls (P < 0.05). Second, the effect of sperm pre‐incubation for 4 h on subsequent in vitro fertilization was assessed using different sperm genetic backgrounds. The pre‐incubation of sperm did not decrease the embryonic development regardless of the breed of the sperm. A milder heat shock (40.0°C) effect on parthenotes (Hol and JB) and IVP embryos were evaluated. JB parthenotes showed developmental arrest after Day 4, and the rate of development to the blastocyst stage decreased by heat shock, but not in Hol parthenotes. Heat shock decreased developmental competence after cleavage of IVP embryos regardless of genetic background. The thermotolerance of IVP embryos would be controlled by both maternal and paternal factors but genetic involvement was still unclear. Further evaluation is needed to reveal the genetic contribution to thermotolerance.     

Effect of relaxin on the motility, acrosome reaction and utilization of glucose of fresh and frozen-thawed bovine spermatozoa

The study was conducted to investigate the effect of relaxin on motility, acrosome reaction (AR), viability and utilization of glucose in fresh and frozen‐thawed bovine spermatozoa. Both semen samples were washed twice through centrifugation (5 min at 600 g), and preincubated for 1 h at 39°C for swim up. The swim‐up separated spermatozoa were resuspended in a sperm Tyrode's albumin lactate pyruvate (Sp‐TALP) medium containing 0 (control) and 40 ng/mL porcine relaxin and incubated for 0–6 h. Sperm motility was determined on the basis of movement quality examined by a phase contrast microscope. Sperm viability and AR were evaluated by using the triple staining technique. The incorporation and oxidation of ¹⁴C‐glucose was assessed by a liquid scintillation counter. Motility was improved (P < 0.05) in both fresh and frozen‐thawed spermatozoa by the addition of relaxin to the Sp‐TALP medium, whereas relaxin showed no significant effect on viability in either fresh or frozen‐thawed spermatozoa. The percentage of AR increased (P < 0.05) when fresh or frozen‐thawed spermatozoa were incubated with relaxin. In contrast, the incorporation and oxidation of ¹⁴C‐glucose increased (P < 0.05) in both kinds of spermatozoa incubated with relaxin. Thus the results demonstrated that the addition of relaxin to the Sp‐TALP medium increased the motility, AR and utilization of glucose in fresh and frozen‐thawed bovine spermatozoa.     

Vitamin E and organic selenium enhances the antioxidative status and quality of chicken semen under high ambient temperature

1. The effects of supplemental dietary vitamin E and organic selenium (Se), and their combination, on improving semen quality characteristics and antioxidative status were investigated in cockerels exposed to high ambient temperature.

2. A total of 36 Egyptian local cross males, 40 weeks old, were housed individually in cages in an open-sided building (average daily temperature ranged from 33 to 36°C and relative humidity from 60 to 70%). Birds were divided randomly into 4 experimental treatments (n?=?9 each): (1) control (basal diet without any supplementation with vitamin E or Se); (2) vitamin E (basal diet +200?mg α-tocopherol acetate/kg diet); (3) Se (basal diet +0·3?mg organic Se/kg diet); and (4) vitamin E?+?Se (basal diet +200?mg α-tocopherol acetate/kg diet +0·3?mg organic Se/kg diet).

3. Under heat stress conditions, inclusion of vitamin E and/or organic Se in the diets enhanced the semen quality traits, including the spermatozoa count and motility, and reduced the percentage of dead spermatozoa.

4. A combination of 200?mg/kg vitamin E with 0·3?mg/kg organic Se reduced the thiobarbituric acid reactive substance concentration in seminal plasma samples to about 28% of the controls; and also enhanced the seminal plasma glutathione peroxidase activity by two-fold compared with controls.

5. It was concluded that dietary vitamin E in combination with organic Se has a synergistic effect in minimising lipid peroxidation and improving the antioxidative status in seminal plasma of the domestic fowl, which probably translated into enhanced spermatozoa count, motility and reduced percentage of dead spermatozoa under heat stress conditions.     

In vitro formation of holes on the inner perivitelline layer of quail ovum by chicken spermatozoa

The aim of the present study was to examine whether chicken semen can be substituted for quail semen to conduct in vitro experiments on fertilization. Chicken spermatozoa was incubated with the inner perivitelline layer (IPL) isolated from the largest follicle in the quail ovary under in vitro conditions. The perforation of chicken and quail spermatozoa were assessed by counting the number of all visible holes in the pieces of IPL. No difference was found in the number of holes formed by the chicken sperm and quail IPL interaction compared with that between intraspecies. In addition, the number of holes in the IPL was significantly increased with the increase in sperm concentration from 1 × 10⁶ to 8 × 10⁶ sperm/mL (P < 0.05). Interestingly, after treatment of chicken spermatozoa with 2.5 mg/mL of the solubilized quail IPL followed by incubation with the intact chicken IPL, the number of holes in the intact chicken IPL was significantly decreased as compared with that of spermatozoa treated without the solubilized IPL (P < 0.05). This indicates that sperm receptors in the solubilized quail IPL and binding ligands on the chicken spermatozoa would find each other and bind, forming complexes and these complexes blocked the interaction between chicken spermatozoa and intact chicken IPL. These results show that: (i) chicken spermatozoa possess the penetrability into quail IPL; and (ii) a high degree of affinity via the receptor interactions exists between chicken spermatozoa and quail IPL. Therefore, it appears that the substitution of chicken semen for quail semen is possible to use as an in vitro technique to examine the sperm‐IPL interaction during fertilization in quail.     

Antioxidant supplementation,effect on post‐thaw spermatozoan function in three sturgeon species

High levels of reactive oxygen species (ROS), which may be associated with reduced sperm quality, can be detected during cryopreservation of sperm of some species. Our objective was to investigate whether the addition of antioxidants to cryopreservation extenders influenced post‐thaw sperm characteristics and fertility in Acipenser dabryanus, Acipenser sinensis and Acipenser baerii. Prior to freezing, sperm samples were diluted with a base extender as control or in extender supplemented with catalase (CAT), glutathione (GSH), cysteine (NAC), ascorbic acid (VC) or their paired combinations. Protective concentrations of CAT, GSH and VC in the three species were 25 U/ml, 0.25‐0.5 mg/ml and 0.5 mg/ml, respectively. Cysteine showed no protective effect against ROS. The addition of CAT, GSH and VC positively affected either acrosome or membrane integrity of post‐thawed sperm in the three species, as well as spermatozoan motility in A. sinensis. The combination of antioxidants did not show a positive synergistic effect. This study suggested that the use of antioxidants in the cryopreservation of sturgeon sperm has potential to decrease intracellular ROS, and consequently preserve acrosome and membrane integrity, as well as spermatozoan motility.     

Retraction of articles by Dr M. Aramli

The below articles published online on Wiley Online Library ( wileyonlinelibrary.com ) have been retracted by agreement between the submitting author, Mohammad Sadegh Aramli (2015, 2015a, 2015b), the Editor‐in‐Chief, Michel Goldberg, and Blackwell Verlag GmbH. After a thorough investigation, there is strong evidence to indicate that the peer review of these papers was compromised. The identities of the reviewers were unable to be verified, and it is believed that these papers were accepted based on recommendations from reviewers not suitably qualified. REFERENCE Aramli, M. S. (2015). Study of enzyme activities and protein content of beluga (Huso huso) semen before and after cryopreservation. Journal of Animal Physiology and Animal Nutrition, 99, 13–16. https://doi.org/10.1111/jpn.12199 Aramli, M. S., Habibi, E., Aramli, S., & Nouri, H. A. (2015b). Determination of some blood and seminal plasma ions in the beluga, Huso huso (Linnaeus, 1758). Journal of Animal Physiology and Animal Nutrition, 99, 17–20. https://doi.org/10.1111/jpn.12225 Aramli, M. S., Kalbassi, M. R., & Gharibi, M. R. (2015a). Effects of multiple collections on spermatozoa quality of Persian sturgeon, Acipenser persicus: Motility, density and seminal plasma composition. Journal of Animal Physiology and Animal Nutrition, 99, 66–72. https://doi.org/10.1111/jpn.12212     

Effects of long‐term in vitro exposure of ejaculated boar sperm to zearalenone and α‐zearalenol in sperm liquid storage medium

The effects of in vitro exposure of porcine spermatozoa to zearalenone (ZEN) and α‐zearalenol (α‐ZOL) were studied by evaluating several parameters of an in vitro fertilization (IVF) system. For this purpose, boar spermatozoa cultured with semen storage medium containing 0 (control), 10 and 1000 µg/L of ZEN and α‐ZOL for 1 week at 5°C were used for IVF of in vitro matured oocytes. Overall, there were no significant differences in the rates of total penetration, monospermic fertilization, and polyspermic fertilization of oocytes inseminated with spermatozoa from the different groups. Similarly, ZEN and α‐ZOL at 10 and 1000 µg/L did not have detrimental effects on the cleavage and development to blastocysts of oocytes after in vitro fertilization. Although the motility, viability, and plasma membrane integrity of spermatozoa significantly decreased after 3 weeks of storage compared to non‐stored spermatozoa (P < 0.05), ZEN and α‐ZOL at the evaluated concentrations did not exert detrimental effects on the above parameters, even after 3 weeks of storage. These results indicate that prolonged exposure of boar spermatozoa to ZEN and α‐ZOL up to 1000 µg/L under reduced metabolic conditions does not affect their in vitro function.     

Quality and Fertilizing Ability In Vivo of Sex‐Sorted Stallion Spermatozoa

Little information is available on the quality of stallion spermatozoa after sex sorting. The objectives of the present study were to assess the quality of sex‐sorted stallion spermatozoa and determine its fertilizing ability after hysteroscopic low dose insemination. Ejaculates from four stallions were collected and sorted by a MoFlo SX^® flow cytometer/sperm sorter. Before and after sorting, spermatozoa were evaluated for motility by Computer Assisted Sperm Analysis, viability (SYBR 14‐propidium iodide), mitochondrial function (JC‐1) and acrosomal status (fluorescein isothiocyanate Pisum sativum agglutinin conjugated). A fertility trial was carried out on four mares (seven oestrous cycles) by hysteroscopic insemination, depositing 5 × 10⁶ X‐bearing spermatozoa. Sex sorting resulted in a significant decrease (p < 0.001) in all motility characteristics. Sperm viability and percentage of spermatozoa with functional mitochondria were not affected by the sorting process, while the percentage of reacted spermatozoa was higher (p < 0.01) for non‐sorted than sorted spermatozoa. Pregnancy rate was 28.6% (2/7) after low dose hysteroscopic insemination. Only one pregnancy was carried to term with the birth of a healthy filly. In conclusion, despite the reduction in sperm motility, sex sorting did not impair stallion sperm viability and mitochondrial activity immediately post‐thaw; moreover, the sexed spermatozoa retained the ability to fertilize in vivo.     

On the Species Specificity of Sperm Binding and Sperm Penetration of the Zona Pellucida

Sperm binding and sperm penetration of the zona pellucida (zp) are regarded as species‐specific. In this investigation, the interactions between bovine oocytes and porcine, respectively, equine spermatozoa have been studied under in vitro conditions and compared with the normal in vitro fertilization of bovine oocytes by bovine sperm. Surprisingly, many of the heterologous spermatozoa adhered firmly to the bovine oocytes and could not be removed by intense washing. On average, more than 100 boar or equine spermatozoa were bound to the zp of bovine oocytes. Electron microscopic studies clearly demonstrated that porcine sperm attached to the zona and underwent the acrosome reaction. Equine spermatozoa displayed a similar binding affinity, but unlike the porcine spermatozoa even penetrated the zp and were taken up into the oocyte after a longer period of co‐incubation. Considering these new results the dogma of a strict species specificity of sperm zona interactions under in vitro conditions has to be reconsidered.     

Motility Characteristics and Fertilizing Capacity of Boar Spermatozoa Stained with Hoechst 33342

Flow cytometry sorting of spermatozoa using fluorescence dye Hoechst 33342 is the only effective sex selection methodology validated in numerous laboratories. This study was carried out to determine the effect of Hoechst 33342 on the motility and fertility of stained boar spermatozoa. Experiment 1 evaluated motility parameters (percentage of motile spermatozoa, velocity, angularity and oscillation) of boar spermatozoa stained with Hoechst 33342 by a computer‐aided sperm analysis (CASA) instrument. Spermatozoa (30 million/ml) were divided into five treatment groups and stained during 1 h at 35°C with 9, 18, 27, 60 and 90 μM of H33342. There were no differences in sperm motility patterns nor percentages of motile spermatozoa incubated in the presence of 9, 18 or 27 μM. Percentage of motile spermatozoa and motility parameters decreased significantly (p < 0.05) at 60 μM of Hoechst 33342. Spermatozoa were immotile at concentration of 90 μM. In experiment 2, pregnancy rates, farrowing rates and litter size from sows (n = 275) artificially inseminated (AI) with either Hoechst 33342 stained (27 μM) or unstained (control) spermatozoa were determined. Sows inseminated with stained spermatozoa had no significant lower pregnancy rate (88.33%) as compared with controls (90.32%). Staining neither affected farrowing rates (85.0 vs 87.7%) nor total number of piglets born (10.56 ± 0.32 vs 10.47 ± 0.24, stained and controls, respectively). No phenotypical abnormalities were registered among the newborn piglets. The data suggest that incubating spermatozoa with Hoechst 33342 at levels required for X‐ and Y‐bearing chromosome sperm sorting, does not impair sperm viability or their fertility after AI.