ALBERT

Notes: Since the first work of Blaxter in 1953, fish sperm cryopreservation has been attempted on about 30 marine species. The present paper reviews the techniques used and the results published in these species. Particular attention is paid to the handling procedure of sperm before freezing, the problems of semen ageing and semen contamination with urine. The quality of frozen–thawed semen was evaluated using previously standardized biotests, such as a two-step motility activation technique adapted for the different species and fertilization assays using a discriminating insemination technique. Most extenders used in marine fish are saline or sugar solutions. From the investigated cryoprotectants, dimethyl sulphoxide (DMSO) generally leads to the best results. Cooling rates range from 8 °C to 99 °C min−1; the thawing rate is generally high. Compared with freshwater species, a high percentage of spermatozoa survives cryopreservation. Therefore, and because of the simplicity of the techniques, the cryopreservation of marine fish sperm is suited for application in aquaculture.

Notes: Morphological investigations on the changes in flagellar beating was carried out on native (taken from the milt) and thawed sperm of the Siberian sturgeon Acipenser baerii (Brandt). Immediately after activation, the pattern of flagellar wave formation and distribution was the same in native and thawed sperm but, after 27–42 s, depending on the samples, the thawed flagella showed asymmetric and poorly developed waves. The swimming trajectories recorded during 1-s exposure were much shorter in thawed than in native sperm after 26–28 s motility. In native sperm, the flagella remained in the same axis as the head during the entire motility course, while the head of thawed sperm showed a right angle after 47 s. It is concluded that the freezing/thawing procedure induces some alteration in the dynamics of flagellar beating in many sperm, but these sperm still show progressive displacement. Therefore, the change in morphology of the flagellum during motion is a parameter that should be taken into account in the evaluation of the impact of various treatments on sperm motility.

Notes: Seabass Dicentrarchus labrax sperm concentration was high (up to 60 × 109 spz ml−1) but decreased significantly at the end of the reproductive season (mid-March) in monthly sampled fish. The spermiation period may be shortened by frequent stripping. Sperm can be prediluted up to 1: 128 in non-activating medium without loss of initial motility and motility duration. Immediately after activation by transfer to sea water, all the spermatozoa were motile for 10 s and then the number of motile cells decreased progressively but sharply to zero, so that the duration of sperm motility was very short (40 s). As a consequence, the fertility of seabass sperm decreased exponentially after 10 s following sperm activation and was zero by 1 min. The sperm requirements for optimal fertilization were c. 66 000 spermatozoa per egg. Scalingup of the experimental insemination procedure yielded better fertilization rates while conserving the individual differences due to the breeder pairs.

Notes: Ninety to 100% of paddlefish Polyodon spathula were motile just after transfer into distilled water, with a velocity of 175 μm s-1, a flagellar beat frequency of 50 Hz and motility lasting 4–6 min. Similarly, 80–95% of shovelnose sturgeon Scaphirhynchus platorynchus spermatozoa were motile immediately when diluted in distilled water, with a velocity of 200 μm s-1, a flagellar beat frequency of 48 Hz and a period of motility of 2–3 min. In both species, after sperm dilution in a swimming solution composed of 20 mM Tris–HCl (pH 8·2) and 20 mM NaCl, a majority of the samples showed 100% motility of spermatozoa with flagella beat frequency of 50 Hz within the 5 s following activation and a higher velocity than in distilled water. In such a swimming medium, the time of motility was prolonged up to 9 min for paddlefish and 5 min for sturgeon and a lower proportion of sperm cells had damage such as blebs of the flagellar membrane or curling of the flagellar tip, compared with those in distilled water. The shape of the flagellar waves changed during the motility phase, mostly through a restriction at the part of the flagellum most proximal to the head. A rotational movement of whole cells was observed for spermatozoa of both species. There were significant differences in velocity of spermatozoa between swimming media and distilled water and between paddlefish and shovelnose sturgeon.

Notes: Au cours des quinze dernières années, les spermatozoïdes et le liquide séminal des poissons téliostéens ont fait l'objet de nombreux travaux dans le but de comprendre les processus mécaniques et chimiqes régulateurs de la motilité. Les spermatozoïdes de truite et de carpe ont été plus articulièrement étujiés; leur flagelle est du type classique “9 + 2” doublets de microtubules auquels sont associés des bras internes et externes de dynéine. Les battements flaellaires sont engendrés par des cycles d' accrochage/ décrochage des bras de dynéine d'un microtubufe au microtubule adjacent, l'énergie provenant de I'hydrolyse de l'ATP. Une protéase couplée à une ligase interviendrait également. Les spermatozoïdes subissent une altération structurale lors de l'émission dam l'eau douce qui ne se produit pas dans la solution saline d'activation; cependant, dans les deux cas, la mobilité des spermatozoïdes est brève, 30sec. chez la truite et une minute chez la carpe. Leur trajectoire est circulaire; la fréquence des battements flagellaires, élevée lors du déclenchement de la mobilité (50–60 Hz), décroît ensuite jusqu'à 10–20 Hz: on constate alors l'arrêt du déplacement des têtes spermatiques bien que les flagelles des sermatozoïdes de carpe continuent leurs battements à une fréquence faible (〈 10 Hz). La vitesse de déplacement diminue parallèlement à la fréquence et la distance moyenne parcourue est de 3 mm chez la truite et 5 mm chez la carpe. La mobilité des spermatozoïdes de truite et de carpe est influencée par l'environnement ionique. Une forte concentration en ion K+ chez la truite et une pression osmotique élevée chez la carpe, inhibent la mobilité. D'autres ions tels que H+ Ca2+ Mg2+ interviennent également dans la régulation du mouvement. Lors de l'initiation du mouvement des spermatozoïdes de truite, I'activité de l'adénylate cyclase et la concentration en AMPc augmentent. L'AMPc serait donc un facteur du déclenchement de la mobilité, il entraînerait la phosphorylation d'une protéine 15 Kd par l'intermédiaire d'une Tyrosine protéine kinase et interviendrait aussi in vivo dans l'acquisition de la mobilité. Lorsque les spermatozoïdes de carpe ne sont pas mobiles dans la solution d'activation, ils peuvent subir une “maturation in vitro” par incubation dans un milieu riche en potassium et de pression osmotique élevée. Aprés l' initiation du mouvement dans une solution d'activation, la quantité d'ATP diminue raidement dans les deux espèces. L'ATP se régénère spontanément dans les uinze minutes qui suivent l' arrêt du mouvement chez la truite. Chez la carpe il n'y a pas de restauration de l'ATP dans la solution saline d'activation mais elle est possible en replasant les spermatozoïdes en condition d'immo-bilisation (forte pression osmotique). II a été montré que des spermatozoides de truite démembranés sont réactivables en présence d'ATPMg2+ et d' AMPc, alors que chez la carpe l'AMPc n'est pas néces-saire.〈section xml:id="abs1-2"〉〈title type="main"〉Summary Spermatozoa motility of trout (Oncorhynchus mykiss) and carp (Cyprinus carpio)In the past fifteen years, teleost spermatozoa and seminal plasma were studied with the aim of understanding the mechanical and chemical processes which regulate motility. Trout and carp spermatozoa were particularly studied; their flaella are classic, with “9+2” microtubule doublets and outer and inner associated dynein arms. Flagellar beating results from hooking/unhooking cycles from the dynein arms of one microtubule to the one adjacent through ATP hydrolysis. A protease coupled to a ligase is probably also involved. Spermatozoa undergo drastic structural change when shed in freshwater but not in an activating saline solution; however, in both cases, motility is short, 30 sec. in trout and one minute in carp. Trajectories are circular; flagellar beat frequencies are high at the beginning of the motility (50–60Hz), and decrease to 10–20Hz: one observes that sperm head stop, whereas flagella of carp spermatozoa continue beating at low frequency (〈 10 Hz). The velocity decreases with frequency; the mean duration of displacement is 3 mm in trout and 5 mm in carp. Trout and carp spermatozoa motility are influenced by the ionic external environment. A high concentration of K+ ions in trout and a high osmotic pressure in carp inhibit motility. Other ions like H+ Ca2+ Mg2+ also interfere with the motility regulation. At the time of trout spermatozoa movement initiation, adenylate cyclase activit and cAMP concentration increase. cAMP is likely a factor involved in the initiation of motility, which may also involve the phosphorylation of a protein 15 Kd through a Tyrosine protein kinase activity. cAMP may also be involved in the acquisition of sperm motility in vivo When carp spermatozoa are immotile in the activating solution, they can undergo a “maturation in vitro” (i.e. the acquisition of the capacity to move) by incubation in a medium with high concentration of K+ and with a high osmotic pressure (400 m Osm/kg). After initiation of movement in the activating solution, intracellular ATP decreases rapidly in the spermatozoa of the two species. ATP regeneration occurs spontaneously within fifteen minutes following the arrest of movement in trout. In carp there is no ATP regeneration in activating saline solution, but replacing sermatozoa in an immobilizing solution (high osmotic pressure) results in a recovery of the intracellular ATP content. It was shown that demembranated trout spermatozoa can be reactivated with ATPMg2+ and CAMP; in contrast, cAMP was not necessary in carp.

Notes: A small urinary bladder attached to the seminal duct in caudal part of the abdominal cavity was registered for the first time in dissected males of tench. The urinary bladder wall was of whitish color and the bladder contained 0.5–2 ml of urine. When collected in the experiment, the tench sperm was white-colored. Spermatozoa density is highly variable due to contamination by urine, and the latter additionally activates spontaneous motility of the spermatozoa. Seminal fluid contains ions such as Na+ (18.4 ± 1.3 mm), K+ (1.9 ± 0.6 mm), Ca2+ (0.6 ± 0.2 mm) and Mg2+ (0.5 ± 0.1 mm), leading to osmolality of 230 ± 82 mOsmol kg−1 depending on the dilution by urine. Urea was detected in urine samples uncontaminated by sperm with an osmolality of 85 ± 58 mOsmol kg−1. Urine also contained high concentrations of ions such as Na+ (30.9 ± 8.9 mm), K+ (4.3 ± 2.9 mm), Ca2+ (0.9 ± 0.5 mm) and Mg2+ (0.6 ± 0.2 mm). The spontaneous sperm activation by urine was up to 100%, but could be prevented by collection in an immobilizing solution. Motility was observed for 90–100% spermatozoa just after their transfer to distilled water or in a swimming medium (SM, 30–45 mm KCl) with a velocity of 120–140 μm s−1. A flagellar beat frequency of 60–70 Hz and forward motility lasted up to 80 s in distilled water, and up to 180 s in SM at room temperature.

Notes: The Siberian sturgeon (Acipenser baeri) and the sterlet (A. ruthenus) were injected with dried sturgeon pituitary (2 mg kg−1), yielding 24 h later respectively 1.71 ± 0.5 and 1.65 ± 0.5 1011 spermatozoa kg−1 body weight. Spermatozoa were best activated with a solution of Tris HC1 50 mM, pH 8.0. The percentage of activated cells was 88 ± 4.4 in A. baeri (n = 5) and 68 ± 19 in A. ruthenus. (n = 5). In A. baeri, immediately after activation, the beat frequency of the flagellum and the mean velocity were in the range of 48–52 Hz and 100–300 μm−1s, respectively. The beat frequency declined to 15 Hz at 30–40 and velocity to 100 μm s−1 at 60 s post-activation. Only a small percentage of the spermatozoa remained motile after 3–4 min. In all cases spermatozoa showed mostly quasi-linear trajectories. Sperm was frozen in liquid nitrogen vapor immediately after dilution 1 v: 1 v in a cryopreservation medium (23.4 mM saccharose, 118 mM Tris-HCl pH 8.0, 20% egg yolk to which 15% DMSO were added). After fast-thawing procedure (25 s at 40°C), the percentage of motile spermatozoa (once activated in 118 mM Tris-HCl, pH 8.0) decreased to 23 ± 8.8 in A. baeri and to 15 ± 11 in A. ruthenus. The fertilizing capacity also decreased: 53 ± 8.3% vs. 89 ± 7.6% in control (P 〈 0.05) in A. baeri and 23 ± 11% vs 53 ± 8.3% in control (P 〈 0.05) in A. ruthenus. The motility pattern of the surviving frozen/thawed spermatozoa was the same as in fresh spermatozoa.