A brief review of the methodology and cryoprotectants in selected fish and mammalian species.

Cryopreservation is a valuable technique used to assist in the genetic improvement of cultured stocks and provide a continuous supply of good-quality semen for artificial insemination. Conserving semen by cryopreservation serves several purposes (e.g. artificial reproductive technologies and species conservation) and is also used in the clinical treatment of human infertility. However, the lifespan of cryopreserved semen is influenced by a range of factors, including storage temperature, cooling rate, chemical composition of the extender, the concentration of cryoprotectant, reactive oxygen species, seminal plasma composition and hygienic control. The choice of cryoprotectant is a vital factor underlying the success of animal semen cryopreservation. In this regard, extensive research has been carried out on various cryoprotectants, such as egg yolk, dimethyl sulfoxide, methanol, ethylene glycol and dimethylacetamide. Recent studies have also described the use of a range of new cryoprotectants for cryopreservation, including compounds of plant origin (soy), amino acids, antifreeze proteins, carbohydrates and cyclodextrins. Moreover, semen cryopreservation and storage require the use of liquid nitrogen or ultralow refrigeration methods for both long- and short-term storage. This review summarizes the general methods used for freezing semen and discusses the use of traditional and newly emerging cryoprotectants (permeable and non-permeable) for the cryopreservation of semen in selected fish and mammalian species.

Effect of dietary antioxidant supplements (selenium forms, alpha-tocopherol, and coenzyme Q10) on growth performance, immunity, and physiological responses in rainbow trout (Oncorhynchus mykiss) using orthogonal array design.

Dietary supplements containing antioxidants play an important role in reducing the risk of peroxidative attack in aquatic animals. In this work, an orthogonal array design (L9: 34) was used to evaluate the effect of four dietary antioxidant supplements on the physiological responses of rainbow trout at three levels. The supplements included different (A) selenium (Se) forms (inorganic, organic, and nanoparticle), (B) Se content (0, 0.3, & 0.5 mg/kg feed), (C) vitamin E (VE) content (0, 100, & 150 mg/kg feed), and (D) coenzyme Q10 (CoQ10) content (0, 10, & 20 mg/kg feed). Fish with an average body weight of 8.35 ± 0.33 g were randomly allocated to different experimental groups. According to the results, the antioxidant supplements included in the diet had no significant effects on the growth performance of fish (P > 0.05). Immunological and antioxidant parameters were mainly improved by the Se form (Nano-Se) and dietary CoQ10 supplementation. In addition, Se form and VE were more effective in digestive enzyme activities and hematology indices in comparison to other dietary antioxidants. Additionally, diets supplemented with nano-Se along with CoQ10 and VE improved fish resistance/stamina against stress. In conclusion, a more effective combination of the four antioxidant supplements was A2/3B2/3C3D3 (i.e., 0.5 mg/kg organic/nano-Se, 150 mg/kg VE, and 20 mg/kg CoQ10), which could mainly improve the physiological responses of rainbow trout.

Hemato-Immunological Responses and Disease Resistance in Siberian Sturgeon Acipenser baerii Fed on a Supplemented Diet of Lactobacillus plantarum.

A feeding trial was conducted to investigate the effects of different levels of dietary Lactobacillus plantarum on hemato-immunological parameters and resistance against Streptococcus iniae infection in juvenile Siberian sturgeon Acipenser baerii. Fish (14.6 ± 2.3 g) were fed three experimental diets prepared by supplementing a basal diet with L. plantarum at different concentrations [1 × 107, 1 × 108 and 1 × 109 colony-forming units (cfu) g-1] and a control (non-supplemented basal) diet for 8 weeks. Innate immune responses (immunoglobulin (Ig), alternative complement activity (ACH50) and lysozyme activity) were significantly higher in fish fed the 1 × 108 and 1 × 109 cfu g-1 L. plantarum diet compared to the other groups (P < 0.05). Furthermore, fish fed on various levels of L. plantarum significantly showed higher red blood cell (RBC), hemoglobin (Hb), white blood cell (WBC) and monocyte compared to those of the control group (P < 0.05). At the end of the feeding experiment, some fish were challenged with S. iniae to quantify the level of disease resistance. The mortality after S. iniae challenge was decreased in fish fed a probiotic. These results indicated that dietary supplementation of L. plantarum improved immune response and disease resistance of Siberian sturgeon juvenile.

Dietary Administration of Lactobacillus plantarum Enhanced Growth Performance and Innate Immune Response of Siberian Sturgeon, Acipenser baerii.

We investigated the effects of Lactobacillus plantarum used as a dietary supplement on the growth performance and innate immune response in juvenile Siberian sturgeon Acipenser baerii. Juvenile fish (14.6 ± 2.3 g) were fed three experimental diets prepared by supplementing a basal diet with L. plantarum at different concentrations [1 × 10(7), 1 × 10(8) and 1 × 10(9) colony-forming units (cfu) g(-1)] and a control (non-supplemented basal) diet for 8 weeks. Growth performance indices were increased in fish fed the 1 × 10(8) cfu g(-1) L. plantarum diet compared to the other groups. There was an increased innate immune response in fish fed the experimental diets. The highest levels of lysozyme activity, total immunoglobulin (IgM) and complement component 3 (C3) were observed in fish fed the diet containing L. plantarum at a concentration of 1 × 10(8) cfu g(-1), but there was no significant difference in the level of complement component 4 (C4) in fish fed the experimental diets or the control diet. The present study underlying some positive effects (growth performance and immune indices) of dietary administration of L. plantarum at a concentration of 1 × 10(8) cfu g(-1) in the Siberian sturgeon.

Effect of freezing rate for cryopreservation of Persian sturgeon (Acipenser persicus) spermatozoa.

This study examined the effect of freezing rate (-10 °C, -15 °C, -20 °C, -30 °C, and -40 °C/min) on motility parameters, rates of fertilization and hatching, ATP content, and indices of oxidative stress including thiobarbituric acid reactive substances and carbonyl derivatives of proteins in Persian sturgeon (Acipenser persicus) sperm. After sampling, sperm was diluted in an extender composed of 23.4-mM sucrose, 0.25-mM KCl, and 30-mM Tris-HCl, pH 8.0, containing 10% methanol and subsequently frozen in a programmable freezer. For postthaw sperm that were frozen at a rate of -40 °C/min, sperm motile duration (134 ± 27.01 seconds), sperm motile percent (60 ± 4.1%), fertilizability (72 ± 8.36% for fertilization rate and 65 ± 7.58% for hatching rate), and ATP content (4.8 ± 0.57 nmol/10(8) sperm) were significantly higher than for sperm frozen at any of the four slower rates (P < 0.05). Moreover, sperm cryopreserved using the fastest freezing rate had significantly lower levels of thiobarbituric acid reactive substances (0.5 ± 0.05 nmol/10(8) sperm) and carbonyl derivatives of proteins (41.3 ± 4.9 nmol/10(8) sperm) than sperm cryopreserved using all other freezing rates (P < 0.05). In addition, there is a significant difference (P < 0.05) between fresh sperm and the recovery of cryopreserved Persian sturgeon sperm using programmable freezing with -40 °C/min being the optimal freezing rate among those tested.

Effectiveness of glucose-methanol extender for cryopreservation of Huso huso spermatozoa.

The present approach was designed to evaluate the methanol-glucose extender effects on sperm cryopreservation in beluga sturgeon, Huso huso. Sperm quality was examined by measuring post-thaw sperm motility and fertilizing rate at hatching stage. We first tested the effect of glucose concentration (0, 0.10, 0.15, 0.20 and 0.30M) in a methanol extender on post-thaw sperm motility. The optimal cryopreservation conditions were found to be 0.2M glucose in the extender. Then, motility and fertilization rates of sperm cryopreserved with 0.2M glucose and 10% methanol (GM) were compared to Tris-sucrose-KCl in 10% methanol extender (TSKM). Additionally, sperm motility and fertilizing ability in relation to 15 and 30min equilibration in GM extender before and after cryopreservation were measured. Higher post-thaw sperm motility duration and percentage as well as fertilization rate were obtained with the GM extender when compared to TSKM extender. Equilibration of sperm in extender did not affect the motility quality of either fresh-diluted or frozen/thawed sperm, while fertilization rate showed a significant decline alone after 30min of post-thaw storage. Our results indicated that the use of a simple extender consisting of 0.2M glucose in 10% methanol can be an alternative cryopreservation method to those previously described for sturgeons.

Effects of dietary ß-glucan on the growth and innate immune response of juvenile Persian sturgeon, Acipenser persicus.

The present study evaluated the effects of different levels of dietary ß-glucan (MacroGard(®)) on growth performance and innate immune parameters in juvenile Persian sturgeon (Acipenser persicus). Fish (20.1 ± 0.8 g) were allocated into 12 tanks (15 fish per tank) and triplicate groups were fed a control diet or diets containing 0.1%, 0.2% and 0.3% ß-glucan. After six weeks of daily feeding (3.0% body weight day(-1)), humoral innate immune parameters (serum immunoglobulin (Ig) levels, lysozyme activity and alternative complement activity (ACH50)) and growth parameters (final weight, weight gain, specific growth rate (SGR), food conversion ratio (FCR)) were examined. Innate immune responses (lysozyme activity and ACH50) were significantly higher in 0.2% and 0.3% ß-glucan fed fish (P < 0.05). Similarly, elevated growth performance (final weight, SGR and FCR) was observed in fish fed 1%, 2% and 3% ß-glucan compared to the control group (P < 0.05). However, dietary ß-glucan had no significant effect on survival rate of fish (P > 0.05). These results indicate that ß-glucan can be considered as a beneficial dietary supplement for improving the immune response and growth performance of Persian sturgeon.

Motility and fertilizing ability of cryopreserved Caspian brown trout (Salmo trutta caspius) sperm: Effect of post-thaw storage time and different sperm-to-egg ratios.

This study was designed to test the effect of post-thaw storage time on sperm motility parameters of Caspian brown trout (n=7). Furthermore, we investigated the effect of sperm-to-egg ratios of 100,000:1, 300,000:1 and 600,000:1 on fertility of cryopreserved Caspian brown semen. Quality was assessed by measuring sperm motility parameters and fertilization rates at the eyed and hatching stages. The percentage of post-thawed sperm motility, curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) were not affected by 60 min of storage, whereas a decrease in straight line velocity (VSL), average path velocity (VAP) and linearity (LIN) were found in cryopreserved semen. Thus, the cryopreserved sperm of Caspian brown trout could be stored up to 60 min without loss of the percentage of sperm motility. The fertilization rate was not affected by 60 min of post-thaw storage and was over 70% for sperm-to-egg ratios of both 300,000 and 600,000:1. To our knowledge, this study is the first to report the high post-thaw fertilization ability of Caspian brown trout semen at a sperm-to-egg ratio as low as 300,000:1. This procedure after scaling up can be recommended for routine Caspian brown trout sperm cryopreservation.

Introducing a new histologic scoring system for iron deposition in liver of thalassemic patients, compared with atomic absorption spectrometry.

Iron deposition in liver is a major finding in thalassemic patients and because of direct iron toxicity to liver it is associated with several consequences for example liver fibrosis. Liver biopsies from 63 patients were evaluated, 40 (63.5%) were male and 20 (36.5%) were female. The mean age of the patients was 8.01 ± 3.7 and the age range was from 1.8 to 15 years. Histologic grading and staging was performed for each case according to modified HAI (Hepatitis Activity Index) system. Iron scoring was performed according to Sindram & Marx and MTK1-3 scoring systems. The mean (SD) dry weight (dw) of liver specimens was 1.34 (0.11) mg (range 0.20 to 3.80 mg). The mean (SD) of hepatic iron concentration was 230.9 (121.2)µmol/g dry weight. The relationship between the variables HIC, HII (hepatic iron index) and all histological gradings of iron (S&M and MTK1-3) was very strong. The relationship between the HIC and staging by HAI method was good. Significant differences were identified between the mean HIC in scores 1&2 of all histological iron scorings (S&M and MTK1-3), but no significant differences identified between mean HIC in other adjacent scores in all histological iron scorings (S&M and MTK1, 2 and 3). New scoring system introduced by us in this study which considered size and density of iron granules as well as zone of iron deposition was very much the same as simple Sindram and Marx classification.

Effect of freezing rate on motility, adenosine triphosphate content and fertilizability in beluga sturgeon (Huso huso) spermatozoa.

Broodstock selection programs are currently underway for sturgeon species. To complement and further these selection programs we need to develop sperm cryopreservation procedures. In the present study, we describe the effects of freezing rate (-10°C, -15°C, -20°C, -30°C and -40°C/min) on gamete quality characteristics (i.e., duration of motility (s), motility percentage (%), ATP content (nmol/10(8) cells), fertilization rate (%), and hatching rate (%)) in beluga sturgeon, Huso huso. After sampling, beluga sturgeon sperm were diluted in an extender composed of 23.4mM sucrose, 0.25 mM KCl, and 30 mM Tris-HCl, pH 8.0 containing 10% methanol and subsequently frozen in a programmable freezer. Sperm frozen at -40°C/min resulted in means for duration of motility (134 s), motility percentage (69%), ATP concentration (4.8 nmol/10(8) cells), fertilization rate (72%) and hatching rate (65%) that were higher (P<0.05) than those for slower cooling rates. Based on our results, -40°C/min was the best freezing rate (among those tested) for cryopreservation of beluga sturgeon sperm.

Effect of a Probiotic Containing Bacillus licheniformis and Bacillus subtilis and Ferroin Solution on Growth Performance, Body Composition and Haematological Parameters in Kutum (Rutilus frisii kutum) Fry.

This study aimed to assess the efficacy of BioPlus 2B, a probiotic containing Bacillus licheniformis and B. subtilis and Ferroin solution on growth performance, body composition and haematological parameters in kutum, Rutilus frisii kutum, fry. The fish were fed dry pellets containing various ratios of probiotics and Ferroin for 60 days after absorption of the yolk sac. At the end of the trial, growth indices (final weight, weight gain, specific growth rate, daily growth rate, food conversion ratio and condition factor), body composition (crude protein, crude lipid, ash and moisture) and haematological parameters [haematocrit (Hct), haemoglobin (Hb), red blood cells (RBC), white blood cells (WBC), neutrophils (NEUTR), lymphocytes (LYM), mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC)] were assessed. Regarding body composition, total protein levels were higher, and ash, moisture and lipid levels were lower in fish receiving the probiotic and Ferroin treatments compared with the control group (P < 0.05). Fish receiving diets supplemented with probiotics and Ferroin solution showed significantly better growth than those fed the basal diet (control). RBC, Hct, Hb, MCV, MCH and LYM were all highest in fish fed probiotic (1.6 × 10(9) CFU/g dry pellet) + Ferroin solution (7 mg/kg dry pellet) + dry pellets. These results indicate that the combination of probiotic and Ferroin solution represents an effective dietary supplement for improving carcass quality, growth performance and haematological parameters in kutum fry.

Motility and fertility of cryopreserved semen in Persian sturgeon, Acipenser persicus, stored for 30-60 min after thawing.

We investigated the effect of storage times of frozen-thawed Persian sturgeon (Acipenser persicus) semen on the duration of sperm motility, percentage of motile sperm, and fertilization and hatching rates of fresh sperm and sperm stored for 0, 30, and 60 min at 4°C post-thawing. Frozen thawed semen analyzed immediately after thawing had similar quality characteristics as fresh semen. For cryopreserved semen stored for 30 min after thawing the characteristics did not differ to fresh semen and cryopreserved semen. For cryopreserved semen stored for 60 min a significant decline in the parameters was observed. Fertilization and hatching rates were not affected by storage times of maximally 30 min of storage.

Effects of short-term storage on the motility, oxidative stress, and ATP content of Persian sturgeon (Acipenser persicus) sperm.

The effective short-term storage of semen is essential when processing multiple sperm samples and when semen must be transported from collection sites to hatcheries for the fertilization of ova, or to laboratories for cryopreservation. In the present study, the spermatozoa of Persian sturgeon (Acipenser persicus) were used to evaluate the effects of short-term storage on quality parameters (the percentage of motile cells and the total period of sperm motility), oxidative stress indices, and the ATP content. Spermatozoa cells exhibited >50% motility during 6 days of storage where the average total duration of sperm motility varied from 376.42 ± 80.86 s initially to 19.28 ± 10.96 s after 6 days. No motile spermatozoa were recorded after 9 days of storage. The levels of oxidative stress indices (TBARS and CP) and antioxidant activity (SOD) increased significantly with the storage time. The ATP content also decreased significantly after 2 days of storage. The results of this study may facilitate successful reproduction management and cryopreservation protocols for this endangered fish.