Male reproductive biology of European hake Merluccius merluccius

The reproductive biology of European hake (Merluccius merluccius) has been studied extensively in the field, but mainly focusing on fecundity regulation in females and its implications for the fishery. The European hake is highly important commercially throughout its geographical range. Because catches have been decreasing since the 1960s, interest in hake as a potential aquaculture species has recently increased. However, for successful domestication of hake, a better understanding of its reproductive biology, including sperm biology, is needed for purposes of broodstock management and also for the development of sperm storage techniques, including cryopreservation. The objectives of this thesis were to assess hake sperm quality, including sperm production characteristics and energetics, and to characterize sperm movement parameters such as the percentage of motile cells, sperm velocity and flagellar beats. Changes of these parameters over time following activation were evaluated qualitatively for fresh hake sperm. The effects of i) salinity of the activation medium, ii) survival in relation to short term storage duration at 4°C, and iii) sperm cryopreservation on sperm motility characteristics, as well as the reliance on the sperm’s energetic content, were evaluated. In many other Gadoid species, drumming muscles are an important component of reproductive behaviour in spawning males: the contraction of these muscles associated to the swim-bladder results in an audible ‘drumming’ sound during the courtship of the females. However, the presence of drumming muscles has never been reported for hake. Mature hake collected for the sperm analyses, as well as mature females and immature individuals were thus dissected to investigate the presence of drumming muscles and the existence in mature males of potential correlations between their morphological characteristics and the sperm motility parameters. Hake sperm were collected from mature males caught during the summer-early autumn waters off western Norway and during the winter-early spring in the Bay of Biscay (France). Sperm quality characteristics were assessed after storage at 4°C for 25 ± 14 h for transportation. The total ATP, ADP and AMP concentrations were measured using high-performance liquid chromatography followed by calculation of the Adenylate Energy Charge (AEC). Computer Assisted Sperm Analysis (CASA) was used to measure a series of head parameters characterizing the sperm swimming performances. The flagellar characteristics of spermatozoa were explored by high resolution video images. Dissections of mature and non-mature European hake males and females were conducted to investigate the presence of drumming muscles. Sperm production characteristics were evaluated for both Norwegian (Nw) and French (Fr) sperm samples: sperm volume in ml (Nw: 3.9 ± 4.0; Fr: 2.6 ± 4.0), spermatozoa concentration (in × 109 spermatozoa / ml) (Nw: 6.6 ± 3.2; Fr: 13.9 ± 5.1), spermatocrit in % (Nw: 80.2 ± 3.3; Fr: 81.8 ± 10.7) and total number of spermatozoa (in × 109) (Nw: 23.5 ± 30.0; Fr: 35.1 ± 36.2). Osmolality (349 ± 28 mOsmol / kg) and pH (7.6 ± 0.1) of French samples were also measured. When sperm was activated with 100% filtrated sea water (100 SW), the percentage of motile sperm, the velocity, the straightness of the movement, the flagellar beat frequency, the wave amplitude, the number of flagellar waves and the linearity of flagellar waves shape were initially at maximum but decreased sharply later. As a result, active sperm motility sufficient to allow the sperm to reach and fertilize the egg is limited to only a brief time period post-activation (< 100 sec). When transferred into 50 % sea water diluted with distilled water (50 SW), the percentage of motile sperm and the velocity of the movement increased initially but subsequently reached a maximum followed by a decline. Sperm were motile for a longer duration (up to ca. 1600 s) when activated with an activating medium of lower salinity (50 SW: 498 mOsmol / kg) compared to 100 SW (998 mOsmol / kg) (ca. 450 s). Sperm storage: initial percentage of motile sperm in 100 SW, velocity and straightness of the movement were at maximum after 0.5 - 1 day storage duration and then decreased gradually to reach their minima after about four days. Further, both the Adenylate Energy Charge (AEC = 0.78 ± 0.07) and the Adenosine triphosphate (ATP = 85 ± 80 nmoles × 109 spermatozoa) content decreased with storage duration (minima reached after ca. two days: 0.20 ± 0.09 and 5 ± 4 respectively). Cryopreservation significantly negatively affected the percentage of motile sperm, with 0 - 76.4 % motile sperm following thawing. Dissections of hake fish demonstrated for the first time that this gadoid species contains drumming muscles. The results indicated that sound production by adult hake males was much more frequent during the spawning season than in the rest of the year, i.e., formed a central component of the mating system in this species. The present thesis represents a first step towards a better comprehension of the male reproductive biology of European hake, for which no studies were published. Basic knowledge on sperm biology, including movement characteristics, cell energy but also the effects of salinity on sperm movement were recorded. Based on these descriptive elements, sperm management methods were tested such as short term storage and cryopreservation techniques. While hake exhibited only low sperm production, this was compensated by the fact that hake sperm were actively motile for a relatively long time. While this thesis has increased our knowledge on the sperm characteristics of hake further research is needed on determining the relationship between sperm quality and fertilization success, including after cryopreservation procedures. The availability of a hake broodstock facility would be beneficial for future studies in determining which sexual characteristics (drumming muscles, sperm quality) influence male reproductive success

Sexual dimorphism of drumming muscles in European hake (Merluccius merluccius)

Dissections of mature and non-mature European hake males and females (N = 142) collected in waters off the western coast of Norway and in the Bay of Biscay (France) in 2004–2006 demonstrate for the first time that this gadoid species contains drumming muscles. There were differences in drumming muscles weight with body length, sex and maturity stage. This study shows that the difference between females and males is primarily manifested during the spawning season, seen both in the French and Norwegian samples. For the mature females the drumming muscles dry weight increases only slightly, if at all, with increase in total length. For mature males there is a corresponding rapid increase. There does not seem to be any consistent difference between the average dry weight of the drumming muscles in immature male and immature and mature female hake of the same length, tested on the Norwegian samples. Our results suggest that male hake, like the males of other gadoids studied, may produce sounds in the context of spawning

Sperm motility in European hake, Merluccius merluccius, and characterization of its spermatozoa concentration and volume, spermatocrit, osmolality and pH

Due to stock declines there is a growing interest in farming of European hake Merluccius merluccius. Thus, knowledge of its sperm biology is of importance not only for purposes of broodstock management, but also for the development of sperm preservation techniques. Hake sperm were collected from mature males caught during the summer-early autumn waters off western Norway and during the winter-early spring in the Bay of Biscay (France). Sperm quality characteristics were assessed after storage at 4 degrees C for 25 +/- 14 h. Average (+/- SD) values for Norwegian and French samples respectively were (i) sperm volume: 3.9 +/- 4.0 and 2.6 +/- 4.0 ml; (ii) spermatozoa concentration: 6.6 +/- 3.2 and 13.9 +/- 5.1 x 10(9) spermatozoa/ml; (iii) spermatocrit: 80.2 +/- 3.3 and 81.8 +/- 10.7%; and (iv) total number of spermatozoa: 23.5 +/- 30.0 and 35.1 +/- 36.2 x 10(9). Average osmolality and pH (+/- SD) of French samples were 349 +/- 28 mOsmol/kg and 7.6 +/- 0.1, respectively. Activation by transfer into full sea water (100 SW) or 10% ovarian fluid in sea water (10 OF) occurred synchronously for virtually all spermatozoa and the percent sperm motile decreased with post activation time. When transferred into 50% sea water diluted with distilled water (50 SW) only a few spermatozoa were activated initially but subsequently reached a maximum percentage of motility followed by a decline. Hake sperm motility declined rapidly to 50% of motility 70 s after activation with 100 SW. Sperm were motile for longer when activated with 50 SW (1570 +/- 295 s) or 10 OF (718 +/- 71 s) compared to 100 SW (317 +/- 121 s). Undiluted hake sperm stored at 4 degrees C up to 10 days retained 10% motility when activated with 100 SW. When cryopreserved, the motility recovery index of the cells at thawing ranged from 0 to 76.4%. These results describe for the first time the sperm traits of European hake following successful cryopreservation, and also show the importance of activation medium on sperm motility. (C) 2010 Elsevier B.V. All rights reserved