Spermatophore Generation Times in Penaeus setiferus, P. vannamei, and P. stylirostris

Spermatophore development times were studied in three species of penaeid shrimp, Penueus setifcrus, P. vannumei , and P. stylirosrris . In the terminal ampoule, the progression of spermatophore formation occurred in 4 stages (I-IV). The duration of each stage varied with species and ablation state. In unablated males, with spermatophores manually or electrically removed, structnrally complete spermatophores containing high numbers of morphologically normal sperm were formed in 2–4 days for P. vannamei , 4–6 days for P. stylirostris and 5–7 days for P. setiferus . Unilateral eyestalk ablation of P. setiferus accelerated spermatophore production time by approximately 2 days, and significantly increased spermatophore weight and sperm count without affecting sperm quality.
Spermatophore removal techniques had little effect on development time in P. vannamei . However, in P. setiferus , new spermatophores were produced faster in males mating naturally than in those males whose spermatophores were removed electrically.     

Egg Water Induced Reaction and Biostain Assay of Sperm from Marine Shrimp Penaeus vannamei: Dietary Effects on Sperm Quality

Reproductive performance was evaluated for sexually mature male Penueus vannamei fed one of four diets: 1) commercial 40% protein shrimp growout diet; 2) 100% squid; 3) 50% squid, 25% bloodworms and 25% Artemia; and 4) starvation. Spermatophores were ejaculated manually from tagged males at stocking and every 12 d thereafter. For each half of the compound spermatophore, weight and sperm count were determined. Four methods were used to assay the sperm quality: 1) gross morphology (GM); 2) trypan blue stain (TB); 3) acridine orange stain (AO); and 4) egg water induced reaction (EW). The reaction induced when P. vannamei sperm are exposed to conspecific egg water is grossly similar to that observed in vivo at the surface of the freshly spawned eggs from naturally mated females. Little correlation was found between spermatophore weight and sperm count. The best correlation was found between GM and AO (r²= 0.859, P < 0.01). Many morphologically abnormal sperm did not stain with TB. For most of the spermatophores evaluated; over 80% of the sperm were morphologically normal, were unstained with TB, and were immediately fluorescing light green when stained with AO. When exposed to egg water, sperm from these apparently good quality spermatophores yielded mixed results. The analysis of dietary effects on spermatophore quality were equivocal. Although a significant decline in body weight, spermatophore weight, and sperm count were observed in the starvation treatment as compared to maturation and squid diets, no significant differences were observed in MY of the sperm quality assays.     

Reproductive Performance of Male Argentine Red Shrimp Pleoticus muelleri Bate (Decapoda, Penaeoidea) in Culture Conditions

Spermatophore and sperm quality of male Argentine red shrimp Pleoticus muelleri held under laboratory conditions was evaluated using compound spermatophore weight, sperm count and percentage of live sperm as indicators. Thirty-seven males of S to 20 g wet weight were held indoors in a 3-m diameter circular fiberglass tank for 45 d. They were fed fresh squid, clam and pelletized diet. Spermato-phores were obtained by manual ejaculations. Four shrimps were re-ejaculated to verify the subsequent spermatophore regeneration. Spermatophore mean weights were significantly lower for 5–10 g males than for 15–20 g males ( r = 0.998) (P < 0.05). There were no significant differences in sperm count and percent live sperm among the different weight classes (5–15 g, 15–20 g, 20–25 g). The sperm count varied from 2.22 to 5.62 million. No change in sperm quality was seen in re-ejaculated individuals. During the first 2 wk of the experiment, the spermatophores exhibited healthy morphology and colour; by the fifth week evidence of spermatophore deterioration was apparent. Despite the degree of melanization, sperm quality was not affected and the high variability in sperm count indicated that the artificial ejaculation is adequate in this species.     

Sperm Quality Over Consecutive Spermatophore Regenerations in the Pacific White Shrimp Litopenaeus vannamei

The reproductive potential of males plays an important role in the productivity of captive penaeids. Males present a large variability in their reproductive potential, and thus the selection of appropriate males for reproduction is highly desirable. The present study compares sperm quality at the beginning of the experiment (baseline values) with variations in sperm quality as a result of consecutive spermatophore regenerations. This was done to evaluate possible predictive criteria for optimal sperm quality based on morphological and biochemical criteria in pond-reared Pacific white shrimp Litopenaeus vannamei males. Sperm quality was similar between left and right spermatophore. Sperm count and proportion of normal sperm increased by 86 and 65% (P < 0.05) respectively, from baseline values to third spermatophore regeneration. Proportion of dead sperm progressively decreased with consecutive regenerations, attaining values 33% lower by the third regeneration, compared to baseline values. This indicates that no decrease in sperm quality occurs in L. vannamei during consecutive regenerations and that the existing spermatophores at stocking of males should be expelled to have higher sperm quality in regenerated spermatophores. Furthermore, this procedure allows selecting individuals with high baseline sperm quality based on significant correlations between baseline and regenerated sperm quality observed in the present work. Baseline values of glucose concentration in the hemolymph were positively correlated to baseline values of sperm count (r = 03, P < 0.05). In contrast, baseline values of several lipids in the hemolymph were negatively correlated to several traits of sperm quality at first sampling (baseline values) or at first regeneration. These results are discussed in terms of nutritional requirements of males and of possible predictive criteria of sperm quality.     

Effect of 17α-Methyltestosterone and 17α-Hydroxyprogesterone on the Quality of White Shrimp Penaeus vannamei Spermatophores

Abstract.— Controlled reproduction of penaeid shrimp requires a better utilization of males by sperm quality monitoring and sperm quality improvement. Spermatophores of white shrimp Penaeus vannamei were improved, in terms of increased sperm count, spermatophore weight, and a reduced incidence of sperm abnormalities by a single injection of 17α-methyltestosterone at 0.01 or 0.1 μg/g body weight. 17α-hydroxyprogesterone did not induce a significant improvement in spermatophore quality. These findings indicate that a steroid injection program should be evaluated as a practical way of improving spermatophore quality in commercial operations.     

Spermatophore production and sperm quality of the river prawn Macrobrachium americanum Spence Bate, 1868 fed with different diets

The effects of different diets on spermatophore production and sperm quality were investigated in the river prawn Macrobrachium americanum. River prawns were cultured and fed with three diets for 244 days: fresh food (50% squid meat, Dosidicus gigas and 50% sardine muscle, Sardinops sagax); commercial pellets (35 Purina®); and a 50:50 mixture of both diets. Spermatophore production was recorded every 24 days on average as the percentage of spermatophores produced per extraction per diet, weight and biochemical composition. Sperm quality was measured as the total number of sperm, the proportion of live/dead sperm and normal/abnormal sperm morphology. There were no significant differences in the mean biochemical composition of M. americanum spermatophores for any of the diets. Biochemical composition was 36.3% protein, 25.8% carbohydrate and 4.6% lipids for all data pooled. The weight of spermatophores and sperm counts was not significantly different among diets, nor were there any differences as a function of the male initial total length (p > .05). Male river prawn reproductive exhaustion was observed as a decline in spermatophore production, weight of the spermatophores and the number of sperm cells per spermatophore, with an increasing proportion of dead and abnormal sperm seen throughout the experiment. The recommended period of maintenance in captivity for male broodstock is less than 115 days. It is recommended to feed broodstock males of M. americanum with commercial pellets because no significant differences were detected with the diets tested; pellets are easier to use, ensuring the same spermatophore production and sperm quality that was obtained with fresh food.     

Spermatophore cryopreservation and artificial insemination of black tiger shrimp, Penaeus monodon (Fabricius)

To develop an appropriate cryopreservation protocol for spermatophores of black tiger shrimp, Penaeus monodon, three cryoprotectants (dimethyl sulphoxide (DMSO), methanol (MeOH) and ethylene glycol (EG)) at two concentrations (5% and 10%) were examined. Artificial implantation of spermatophores was also carried out to assess the fertilizing ability of fresh and post‐thaw spermatophores. Spermatophores were collected during consecutive regenerations (15‐day intervals) and assessed for qualitative and quantitative changes and also for fertilizing ability by implantation. The mean fertilization rate for artificial insemination using post‐thaw spermatophore was 79.9±3.7%, lower than the fertilization rates observed for artificial implantation using fresh spermatophore and natural mating. Mean hatch rates for fresh spermatophore, frozen‐thawed spermatophore and natural mating were 88.8±0.6%, 87.8±0.4% and 88.3±0.5%, respectively; and there was no difference among the three groups. The mean fertilization rate of spermatophores collected during the first stripping was higher (90.6±0.6) than during the second stripping (85.7±2.6), but the mean hatch rate was not different between the two strippings. The highest mean sperm viability (79.7±0.4%) was obtained from DMSO (5%), with no survival observed in the 10% MeOH treatment. Spermatophore weight, total sperm count and percentage of abnormal sperm were not different between spermatophores collected at the first and second stripping. This is the first study to report high fertilization and hatch rates from cryopreserved spermatophore using artificial implantation of spermatophore before spawning.     

Sperm Quality of Pond-Reared and Wild-Caught Penaeus monodon in Thailand1

Sperm quality, as determined by visual examination and by reaction with “egg-water” was not significantly different (P > 0.05) for sperm obtained by electro-ejaculation from ablated or non-ablated pond-reared Penaeus monodon. Nor was sperm quality different between pond-reared and wild-caught prawns. Normal sperm, determined by appearance, ranged from 17.1 to 21.0%, while reactive sperm ranged from 1.5 to 3.0%. There were, however, significant correlations (P < 0.01) between spermatophore weight and prawn weight (r= 0.73, N= 434). Male prawns weighing 4150 g produced on average spermatophores weighing 22.7 mg and containing 0.8 million sperm, while prawns weighing 61-90 g produced on average spermatophores weighing 56.6 mg with 2.5 million sperm. Ablation did not increase spermatophore size or sperm quality, although it significantly increased mortality of ablated males. Male prawns could be re-ejaculated at about weekly intervals with no change in sperm quality. Wild-caught female prawns artificially inseminated with spermatophores from electro-ejaculated males produced normal spawns with 51% average egg fertilization, and 41% nauplii hatch success. Nauplii hatch success following spawning increased from >60% for newly inseminated females to near zero after 30 days post-insemination, indicating spermatophore depletion and/or deteriorated sperm quality during spermatophore storage in the thelycum. The findings of the present study indicate that electro-ejaculation and artificial insemination are relatively simple and practical methods for improving captive reproduction performance of closed-thelycum prawns such as P. monodon, and that pond-reared and wild-caught males produced sperm of similar quality.     

眼柄摘除对红螯光壳螯虾生长、性腺发育及体色的影响

为探究眼柄摘除对红螯光壳螯虾(Cherax quadriarinatus)生长、性腺发育及体色的影响,对6月龄红螯光壳螯虾[体质量(47.47±3.48)g]进行了摘除眼柄处理实验。结果显示,4周后摘除双侧眼柄组红螯光壳螯虾体质量为(84.40±13.41)g,显著高于不摘除眼柄组[(49.63±6.47)g]和摘除单侧眼柄组[(51.47±4.08)g](P<0.05);摘除双侧眼柄组红螯光壳螯虾存活率为60.0%±4.8%,显著低于不摘除眼柄组和摘除单侧眼柄组(分别为94.4%±2.3%和97.2%±2.1%);摘除双侧眼柄组红螯光壳螯虾性腺指数为1.23±0.50,显著高于不摘除眼柄组(0.20±0.06)和摘除单侧眼柄组(0.35±0.08)。利用ImageJ软件对红螯光壳螯虾体色RGB值分析表明,摘除双眼柄组红螯光壳螯虾体色G值和B值(分别为99.26±5.23和98.40±3.58)显著高于摘除单侧眼柄组(59.02±3.85和44.07±4.57)(P<0.05)。结果表明,眼柄摘除能促进红螯光壳螯虾生长和性腺发育,影响体色变化。     

The effect of Ibuprofen on female and male reproduction of the open thelyca marine shrimp,Litopenaeus

A research was designed to explore the effect of ibuprofen (IBU) as an intervening agent for prostaglandin biosynthesis on male and female reproduction of three species of Litopenaeus. Ovarian maturation was studied in L. stylirostris and L. vannamei injected periodically with IBU at 0.01 and 0.1 μg g^?1 body weight (b.w.) and treated for unilateral eyestalk ablation. IBU at 0.1 μg induced a statistically (P < 0.05) higher rate of L. vannamei females with developing ovaries (45 ± 19%) as compared with controls (12 ± 8%) during 5 weeks; L. stylirostris did not activate vitellogenesis under this experimental protocol. IBU was also evaluated for male sexuality in L. occidentalis, L. stylirostris and L. vannamei. Periodic injections of IBU had no effect on sperm counts and spermatophore weights; however, sperm abnormalities in IBU 0.1 μg treated L. occidentalis and L. stylirostris were very low. In L. stylirostris, the renovation of normal spermatophores was remarkably improved in IBU treated males as compared with controls, but in L. vannamei spermatophore deterioration was not affected. The findings of this research support the hypothesis that prostaglandins are involved in penaeid reproduction playing a negative physiological role; however, altered steroidogenic pathways could also be involved.     

Reproductive Quality Evaluation of Male Penueus stylirostris from a Grow-Out Pond

Several male Penucus stylirostris were selected from a 3 ha commercial earthen pond and were individually evaluated for reproductive performance. Indicators measured were compound spermatophore weight, sperm count, and sperm abnormalities.
It was found that spermatophore quality was significantly better for 30–40 g shrimp than for 20–30 g shrimp ( P < 0.05). The higher frequency of abnormalities measured in younger males and the inverse relationship between abnormalities and sperm count indicate that the vas deferens could be the tissue responsible for producing highly abnormal immature semen. It is proposed that male maturation has at least three independently controlled levels of organization: testes maturation, vas deferens maturation, and spermatophore synthesis.
The individual evaluation showed that each male followed a particular response in reproductive quality. Changes in spermatophore weight were not an indicator of sperm density within spermatophores.
Male reproductive tract degenerative syndrome (MRTDS) and male reproductive system melanization (MRSM) did not develop in any shrimp during these experiments.     

Effect of diets on carotenoid content,body coloration,biochemical composition and spermatophore quality in the “red cherry” shrimp Neocaridina davidi (Caridea,Atyidae)

In aquarium industry trade markets, Neocaridina davidi, the “red cherry” shrimp, is one of the most attractive ornamental shrimps due to the striking reddish coloration of females. Five commercial diets formulated for ornamental species were evaluated over a 90‐day period in N. davidi males and females, based on survival, biochemical composition, body weight, carotenoid content and body coloration. Furthermore, histological and histochemical analyses of the male reproductive system were performed to evaluate the spermatophore quality. In all treatments, survival was >80%, and protein content was similar in both female and male shrimps. Lipid content was higher in females and males fed on a diet having the highest lipid level. Carotenoid content was higher and body coloration was brighter in females and males fed on a diet containing the highest carotenoid level. Histological and histochemical analyses of the spermatophore structure and composition showed no differences among diets, suggesting that the five commercial diets are adequate for satisfactory growth and spermatophore quality of N. davidi. The diverse sources and quantities of carotenoids in the highest carotenoid level diet render it more suitable for colour enhancement of this species.     

A3α肽聚糖对凡纳对虾幼体生长及抗病毒感染力的影响

用含不同量的A3α肽聚糖(PG)(0.005%、0.01%、0.1%)的幼体饵料投喂凡纳对虾幼体,另在育苗水体中添加不同水平(0.005mg·mL-1、0.01mg·mL-1、0.05mg·mL-1)PG浸浴并以投喂不添加PG的幼体饵料的实验组作为对照,另设投喂活饵的实验组作参考,分别考察Z1-M1期、M1-PL1期、Z1-PL1期幼体的成活率,M1期、PL1期幼体体长,PL1期幼体体重,另外,PL1期仔虾经7d饲养后,检测体内酚氧化酶(PO)活性,并用白斑综合症病毒(WSSV)料投喂感染。结果显示:与投喂不添加PG幼体饵料组相比,在Z1-M1期,活饵组、0.01%PG添加量的幼体饵料组、0.005mg·mL-1PG的浸浴组幼体的成活率有极显著(P<0.01)提高,而在Z1-PL1、M1-PL1期,各实验组除0.05mg·mL-1浸浴组外均有极显著地(P<0.01)提高;各实验组除0.01mg·mL-1、0.05mg·mL-1PG浸浴组外,M1期幼体体长、PL1期幼体体重也有显著(P<0.01)增长,且各实验组PL1期幼体都有极显著(P<0.01)增长;各实验组仔虾PO活力均有极显著(P<0.01)提高;各实验组除0.1%PG添加量的幼体饵料组外,感染WSSV后仔虾的存活率有不同程度地提高。     

切除单侧眼柄对不同体质量养殖斑节对虾卵巢发育及产卵的影响

在盐度31、水温28~29℃下,进行了4次卵巢诱导试验,研究了切除单侧眼柄对体质量56.4~105.4g的养殖斑节对虾卵巢发育和产卵的影响及卵巢发育、产卵与体质量之间的关系。结果显示,切除单侧眼柄诱导养殖斑节对虾卵巢发育成熟,成功率(y)与雌虾体质量(x)显著相关(P0.05),雌虾体质量越小,诱导成功率越低,回归方程为:y=133.92lnx-535.63(r2=0.9297)。养殖斑节对虾眼柄手术后,最快10d产卵,部分雌虾能重复产卵。产卵量(y)与雌虾体质量(x)显著正相关(P0.05),雌虾体质量越小,产卵量越小,回归方程为:y=122.22lnx-472.2(r2=0.9738)。雌虾累积死亡率、卵子直径和卵子孵化率与雌虾体质量无显著相关性(P0.05)。建议用体质量80g以上的养殖斑节对虾雌虾作为亲虾。     

Electrical stimulation of spermatophore extrusion in the freshwater yabby (Cherax destructor)

A technique for collecting spermatophores from male yabbies (Cherax destructor) is described. This technique consists of applying an electrical stimulus of between 5 and 10 mA and 40–50 Hz around the coxa of the fifth walking leg, and was found to have a success rate of 76% in 100 males tested. Furthermore, this approach enabled the collection of spermatophores from intersex individuals, indicating that intersex animals have the ability to function as males.

Using this technique, it was shown that spermatophore development is correlated with water temperature and that it is possible to elicit spermatophores from males in the non-reproductive season simply by raising water temperatures. The ability to non-invasively collect spermatophores from yabbies will facilitate the development of artificial fertilization techniques in this species.     

Experimental broodstock diets as partial fresh food substitutes in white shrimp Litopenaeus vannamei B.

In the first experiment, conducted in a research facility, Litopenaeus vannamei broodstock were fed either a 100% fresh food control treatment (FRE, consisting of frozen squid, oyster, mussel and enriched Artemia biomass in a 2.3:1.4:1.3:1 dry matter ratio) or one of the two treatments in which 50% [dry matter (DM)] of the fresh food was substituted with experimental artificial diets: a dry diet based on freeze-dried Artemia biomass (ART) and a control dry diet (CON). In the second experiment, conducted in a commercial hatchery, shrimp broodstock were fed either a fresh ration (FRE, consisting of frozen squid, polychaetes and enriched Artemia biomass in a 2.5:1.5:1 DM ratio) or the same experimental artificial diets (ART and CON) replacing 50% of the DM by elimination of polychaetes and Artemia biomass. In experiment 1 treatments CON and ART produced better results ( P =0.05) than treatment FRE in terms of spawn performance and egg production per female. In experiment 2 no differences were detected among treatments FRE and CON whereas treatment ART performed better ( P =0.05) in terms of spawning, egg production per female and spermatophore quality. Broodstock survival and offspring quality did not differ between treatments in either experiment.     

ARTIFICIAL REPRODUCTION OF Penaeus japonicus BATE AS A BASIS FOR THE MASS PRODUCTION OF EGGS AND LARVAE1

Penaeus japonicus Bate is absent on the Italian coasts, but it is considered a very promising species for national aquaculture. To develop the shrimp-culture on a commercial basis, a convenient method for producing eggs and larvae of Penaeus japonicus was studied. A stock of 156 females, age class 1+ and F₃, underwent unilateral eyestalk ablation and they were stocked with males in three plastic tanks (bottom area 14 m²) at a density of 25 individuals/m² and a sex ratio of 1:1. Conditions of environment and management consisted of: temperature 21±3°C; natural light and photoperiod; total exchange of sea water every day; feeding on mussels. A month after eyestalk ablation the females were inspected every 7–10 days; mature females were transferred to the spawning tanks. Females with well developed gonads averaged 25.0% of the population of females at every inspection; 51.3% of spawners were impregnated with spermatophores; others were inseminated artificially by the method of introducing spermatophores into the thelycum. In six inspections (carried out during a period of 89 days), 3,028,525 eggs were released; a female released 17,500 eggs on the average (maximum was 123,000 eggs, minimum 4,340). The mean rate of fertilized eggs was 52.7%—67.7% from naturally inseminated females and 7.5% from the artificially inseminated ones. The mean hatching rate was 30.9%—40.1% from natural insemination and 3.3% from artificial insemination. Total amount of viable nauplii was 820,550.     

Unilateral eyestalk ablation improves molting frequency and reproduction in Macrobrachium amazonicum females

This study has investigated the influence of unilateral eyestalk ablation on the survival, molting frequency, fecundity, reproductive output, and fertility of M. amazonicum females. We divided 96 females into with ablation and without ablation groups with six replicates. Water quality, growth, survival rate, intermolt period, molts number, and ovigerous females over a 90-day period were monitored. Unilateral eyestalk ablation reduced intermolt period by 30% and increased molts number, ovigerous females, and reproductive output. Our findings indicate that unilateral eyestalk ablation positively influences reproduction in M. amazonicum and is appropriate for females of this species having a body length of ≥ 70 mm.     

Effect of Water Temperature on Reproductive Tract Condition of Penaeus setiferus Adult Males

Abstract— This study was conducted to determine the effect of water temperature on male reproductive tract degenerative syndrome (MRTDS) and male reproductive system melanization (MRSM) in Penaeus setiferus , and to evaluate the effect of water temperature on spermatophore regeneration time and sperm quality in electrically ejaculated adult males. Variation in sperm quality, gonadosomatic index, and histological changes in the reproductive system were used to assess reproductive tract changes in the first experiment. Sperm quality and regeneration time were measured before and after successive regenerations in the second experiment. Sperm quality was stable when shrimp were held at 26 C for 30 d but was reduced when shrimp were held at 30 C. Reproductive tissues of 20% of the shrimp held at 30 C were melanized to some degree but tissue melanization was found in only 2.5% of the shrimp held at 26 C. Spennatophore regeneration time was also affected by temperature. Average times for first spermatophore regeneration were 192 h at 25 C, 152 h at 30 C, and 144 h at 33 C. Sperm quality of regenerated shrimp was not affected at 25 C but was reduced for regenerated animals held at 33 C. Based on these results, we recommend a water temperature between 25 and 27 C to obtain adequate spermatophore regeneration and to maintain healthy male P. setiferus broodstock for at least 30 d.     

褐藻酸钠裂解物对冷冻南美白对虾品质的影响

以褐藻酸钠为原料,采用过氧化氢氧化法制备褐藻酸钠裂解物,分析了裂解物对南美白对虾Penaeus vannamei虾仁质量的效果,并研究了对虾仁冷冻后品质的影响.结果表明,褐藻酸钠裂解物中的分子量大于6 kDa的裂解物HDX-A能有效改善冷冻后水分流失,虾仁经1％的浸泡液处理1h后进行冻藏不同时间后再解冻,HDX-A能够有效地阻止水分流失,从而保持虾仁水分且随时间的延长其保持能力并不减弱,而且随浸泡溶液浓度的增加效果增强.HDX-A主要是通过增加自由水含量而避免了冷冻后虾仁水分和营养流失.HDX-A还可有效地降低冷冻虾仁的蒸煮损失,1.5％处理的样品蒸煮损失最低,整体上蒸煮损失低于20％,120d时仅为15.6％左右,显著低于对照(P＜0.05).HDX-A可通过降低冷冻虾仁水分流失改善了粘弹性和破断力等流变学特性,提高了产品品质,可作为无磷品质改良剂替代复合磷酸盐.