Changes in Atlantic salmon (Salmo salar) sperm morphology and membrane lipid composition related to cold storage and cryopreservation

The cold storage and cryopreservation of semen decrease sperm quality. Morphological and biochemical analyses of spermatozoa provide valuable information for the optimization of storage protocols to obtain a sufficient number of spermatozoa for in vitro fertilization. The aim of this study was to evaluate the morphology and lipid composition of Atlantic salmon (Salmo salar) spermatozoa after storage at 4 °C and cryopreservation. Semen samples were obtained by stripping. One aliquot was stored at 4 °C for 7 days, and another aliquot was cryopreserved. The morphology and ultrastructure were analysed using electron microscopy. The lipid composition was analysed by gas chromatography and a commercial kit. After cold storage, the mitochondrion was the most affected component; however, plasma membrane rupture and detachment of the flagellum were also observed. Morphological abnormalities were greater in cryopreserved spermatozoa. The head and mid-piece were dehydrated, sperm membranes were vesiculated, and alterations of mitochondria were observed. After cold storage and cryopreservation, there were less polyunsaturated and omega-3 fatty acids. Furthermore, there was an increase in saturated fatty acids and decrease in cholesterol concentration after cryopreservation (P < 0.05). Based on the results, cryopreservation drastically damaged sperm membranes; the cryogenic damage was associated with membrane lipid composition alterations. The sperm membranes were affected less by cold storage but there was also a decrease of some lipids; therefore, there is a need for improvement in cold storage processes to decrease structural damage of spermatozoa so that semen cryopreservation can be effectively used in the salmon industr

Effects of cryopreservation on cAMP-dependent protein kinase and AMP-activated protein kinase in Atlantic salmon (Salmo salar) spermatozoa: Relation with post-thaw motility

Sperm motility in fish with external fertilization is critical for reproductive efficiency in aquaculture, especially in salmonids. Gamete preservation techniques, such as cryopreservation, however, reduce sperm motility and fertilizing capacity. Very few studies have addressed cryodamage from energetic and cell signalling approaches. In this study, cAMP-dependent protein kinase (PKA) and AMP-activated kinase (AMPK) activities were quantified in fresh and cryopreserved spermatozoa of Atlantic salmon (Salmo salar); and the relation with motility was analysed. Results indicate there was a decrease in membrane integrity and motility in post-thawed spermatozoa compared to fresh samples, however, there was about 30% of cells with intact plasma membrane but incapable of motility. The PKA and AMPK activities were less after cryopreservation, indicating that loss of motility may be related to alteration of these key enzymes. Furthermore, PKA and AMPK activities were positively correlated with each other and with motility; and inhibition decreased motility, indicating there is a functional relationship between PKA and AMPK. The PKA inhibition also decreased AMPK activity, but results from protein-protein docking analyses indicated AMPK activation directly by PKA is unlikely, thus an indirect mechanism may exist. There have been no previous reports of these kinase actions in fish spermatozoa, making these findings worthy of assessment when there are future studies being planned, and may serve as base knowledge for optimization of cryopreservation procedures and development of biotechnologies to improve reproduction efficiency in the aquaculture industr

Structure and function of the Ts2631 endolysin of <i>Thermus scotoductus</i> phage vB_Tsc2631 with unique N-terminal extension used for peptidoglycan binding

Abstract To escape from hosts after completing their life cycle, bacteriophages often use endolysins, which degrade bacterial peptidoglycan. While mesophilic phages have been extensively studied, their thermophilic counterparts are not well characterized. Here, we present a detailed analysis of the structure and function of Ts2631 endolysin from thermophilic phage vB_Tsc2631, which is a zinc-dependent amidase. The active site of Ts2631 consists of His30, Tyr58, His131 and Cys139, which are involved in Zn2+ coordination and catalysis. We found that the active site residues are necessary for lysis yet not crucial for peptidoglycan binding. To elucidate residues involved in the enzyme interaction with peptidoglycan, we tested single-residue substitution variants and identified Tyr60 and Lys70 as essential residues. Moreover, substitution of Cys80, abrogating disulfide bridge formation, inactivates Ts2631, as do substitutions of His31, Thr32 and Asn85 residues. The endolysin contains a positively charged N-terminal extension of 20 residues that can protrude from the remainder of the enzyme and is crucial for peptidoglycan binding. We show that the deletion of 20 residues from the N-terminus abolished the bacteriolytic activity of the enzyme. Because Ts2631 exhibits intrinsic antibacterial activity and unusual thermal stability, it is perfectly suited as a scaffold for the development of antimicrobial agents

Effect of Dietary Inclusion of Leucaena (Leucaena leucocephala) and Banana Flour (Musa cavendishii) on Performance of Laying Hens

ABSTRACT The aim of the present study was to evaluate the effects of Leucaena (Leucaena leucocephala) and Banana flour (Musa cavendishii) on performance of laying hens. Fifty laying hens (3 months of age) were randomly distributed into five experimental groups, each consisting of 10 laying hens. The groups were control (10 laying hens); L6 (Leucaena, 6 g/day (10 laying hens)); L8 (Leucaena, 8.0 g/day (10 laying hens)); L10 (Leucaena, 10 g/day (10 laying hens)); and L12 (Leucaena, 12 g/day (10 laying hens)), in addition, five levels of Banana flour control (10 laying hens); 25% (10 laying hens); 50%, (10 laying hens); 75% (10 laying hens); and 100% (10 laying hens), were assessed respectively. The experimental period lasted from 4 to 8 weeks. The results of this study showed that there were no significant differences between of treatments L6, L8, L10, and L12 for body weight during the first 30 days compared with the control, whereas for weight gain, statistically significant differences were observed between the control compared with the treatments L6, L8, L10, and L12 for days 10, 20 and 30 (p<0.05). Additionally, statistically significant differences were found between different levels of Banana flour for weight gain (g) between the control with the levels 25, 50 75, 100%, respectively for days 20 and 30. In the case of feed intake (g) statistically significant differences were found during day 30 between the control and 100%, also between the control and levels 25, and 75%, respectively. From the results, it can be concluded that the inclusion of Leucaena and banana flour have effects on weight gain, body weight and feed intake of laying hens