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.     

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.     

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.     

Effect of vitamin E on spermatophore regeneration and quality of pond‐reared,black tiger shrimp (Penaeus monodon)

The objective of the present work was to evaluate the effects of different levels of vitamin E in feed on the spermatophore regeneration and quality of male Penaeus monodon. The experiment was carried out with the four following treatments: the basal diet no added vitamin E, the diet added 200, 600 and 1,000 mg/kg respectively. Spermatophore regeneration and quality were evaluated by spermatophore weight, sperm count and spermatophore absence rates, which male P. monodon were extruded spermatophore for feeding 20 and 40 days. In the experiment, the weight of the twice regenerated spermatophore of the males added to the vitamin E group was higher than that of the untreated control group. The weight of the first regenerated spermatophore with the addition of 1,000 mg/kg group was the highest and significantly higher than the control group (p < .05), but there was no significant difference among the three groups with different levels of vitamin E. The weight of the second regenerated spermatophore with the addition of 600 mg/kg group was the highest, followed by 1,000 mg/kg group, both of which were higher than the control group and the addition of 200 mg/kg group. Within the same group, the regeneration spermatophore weight showed overall upward trend as the feeding time, twice regenerate experiment spermatophore weight with added to the vitamin E groups were significantly higher than the initial value (p < .05), but three spermatophore weight of male shrimp at the control group had no significant difference. The sperm quantity and the percentage of normal sperm of the twice regenerated spermatophore of the males with added to the vitamin E group was higher than that of the untreated control group, and those of the addition of 200 mg/kg group was significantly higher than that of control group (p < .05). The total number of sperm and the percentage of living sperm of male shrimp in the experimental group decreased with the increase of vitamin E in the feed. Within the same group, the total number of sperm and the percentage of living sperm of male shrimp with added to the vitamin E groups showed overall upward trend as the feeding time and were significantly higher than the initial value (p < .05), but the control group was slightly down and had no significant difference. Comprehensive sperm weight, sperm quantity and living sperm percentage of three indicators, that adding 200 mg/kg of vitamin E in feed could effectively promote the spermatophore regeneration in the male P. monodon and improve the sperm quantity. The experimental results provide a scientific basis for the breeding of P. monodon.     

Sperm production in the red claw crayfish Cherax quadricarinatus (Decapoda, Parastacidae)

The objective of this study is to evaluate the effect of body size, temperature and annual cycle on sperm production in Cherax quadricarinatus. Sperm count and sperm mortality were estimated, the reproductive system was weighted, and macro and microscopical analysis of the testes and vasa deferentia were conducted. Sperm count and weight of the reproductive system are strongly related to male size, in contrast to sperm mortality. The spermatophore structure presented macro and microscopical differences between sizes. Males higher in size have more adherent spermatophores. This species has a reproductive cycle related to sperm production. Sperm count and weight of the vasa deferentia rise in summer, while the weight of the testes increases in winter. During the spring, the sperm cord presents a higher density than in other seasons. The temperature seems to affect sperm production being 27 and 29 °C the best assayed conditions. The present results are relevant information to obtain the best sperm viability selecting male size, season of sampling and the best temperature for the reproductive stock and future assays of spermatophore cryopreservation for this species aquaculture.     

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.     

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.     

Shipping of Penaeid Broodstock: Water Quality Limitations and Control During 24 Hour Shipments

Shipping trials were conducted with adult Penaeus setiferus to determine the effect of 24 hour closed shipments on water quality, to evaluate methods of reducing water quality deterioration and to maximize packing density (biomass). Other trials were undertaken with juvenile P. setiferus and adult P. vannamei for comparison. The method utilized 8 L seawater chilled to 18–19 C, inside doubled polyethylene bags held in a Styrofoam box (42 × 42 × 23 cm deep). Gaseous O₂ was injected into the water to 8 ppm (for standardization) and into the space above the water. Super-saturation of dissolved oxygen (>20 ppm for 24 hours) had no adverse effect on survival. Initial trials resulted in increased ammonia (from 0.1 to 4–6 ppm NH₄-N), increased carbon dioxide (from <2 to 80–100 ppm), increased temperature (from 18–19 to 25–26 C) and decreased pH (from 8.0 to 6.0–6.4). Increases in shipping density (number or biomass of shrimp per L seawater) further intensified water quality deterioration. Carbon dioxide (with concomitant pH decline) is implicated as the major limiting factor during 24 hour closed shipment. Ammonia accumulation was reduced or totally eliminated with addition of AmQuelΘ (Kordon, Inc, Hayward, California), ² depending on the dosage used. The buffer, Trizma® 8.3 (Sigma Chemical Co, St. Louis, Missouri), ² limited CO₂ buildup and reduced pH decline. Maintenance of low temperature over 24 hours was enhanced with addition of a frozen cold pack placed over the shipping bags. However, the control of ammonia, pH and carbon dioxide by the addition of AmQuel and Trizma did not increase survival, possibly due to toxic effects of the chemicals.     

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.     

中国对虾卵水的特性和精子的应答

研究了中国对虾卵水的采集、保存方法、紫外吸收特性和有效期等方面的内容,并应用卵水研究了交尾期虾、雌虾及产卵期雌虾精荚或纳精囊中精子对卵水的应答开始时间、必要的反应时间等响应卵水的时间特性以及精子的存活期等。结果表明,卵水经液氮保存或先经液氮后转入普通冰柜保持冻结状态７个月后诱导精子激活效力无显著差异。精子在自然温度（１０℃）普通海水中可在１０ｈ内保持对卵水的响应能力。交尾期精子最初响应卵水有５￣１     

Effect of water temperature on the immune response of white shrimp Litopenaeus vannamei to Vibrio alginolyticus

White shrimp Litopenaeus vannamei held in 25‰ seawater at 27 °C or 28 °C were injected with TSB-grown Vibrio alginolyticus at 1 × 10⁴ colony-forming units (cfu) shrimp^− 1 or 1 × 10⁵ cfu shrimp^− 1, and then cultivated onward at water temperatures varying from 20 to 34 °C. Over 24–144 h, mortality of V. alginolyticus-injected shrimp held at 34 °C or 32 °C was significantly higher than that of shrimp held at lower temperatures. In a separate experiment, shrimp held in 25‰ seawater at 28 °C and then cultured onward at 20 to 32 °C were examined for immune parameters at 24–96 h. THC, phenoloxidase activity, respiratory burst, and SOD activity decreased significantly at 24 h after transfer to 32 °C. Shrimp held in 25‰ seawater at 27 °C and then cultured onward at 20 to 34 °C showed a significant reduction in phagocytic activity and clearance efficiency for V. alginolyticus at 24 h after transfer to 34 °C. It was concluded that transfer of shrimp from 27 or 28 °C to higher temperatures (32 and 34 °C) reduced their immune capability and decreased resistance to V. alginolyticus infection.     

Sperm capacitation of the shrimp Litopenaeus vannamei

Litopenaeus vannamei is one of the most important species of farmed shrimp. The females have an ‘open’ thelycum. Mating is accomplished by attaching the male spermatophore onto the surface of the thelycum 4–6 h before spawning. During this period, sperm may have to undergo morphological changes associated with a capacitation process that has been described for other shrimp species. The objective of this research was to extend research on sperm capacitation in L. vannamei by ultrastructural and biochemical means. The sperm of L. vannamei were divided into those freshly prepared from the spermatophore (S‐sperm), extracted from the male gonopores, and those extracted from the female thelycum (T‐sperm). Under transmission electron microscopy, ultrastructural differences were detected between the S‐ and the T‐sperm in the nuclear material, the filamentous meshwork and the cytoplasmic particles. Under scanning electron microscopy, the difference was observed in the cap and spike regions. Immunofluorescence using confocal microscopy to detect tyrosine phosphorylated proteins revealed different distribution patterns between S‐ and T‐sperm. The location of phosphorylation activity changed from the spike in S‐sperm, to the filamentous meshwork in the T‐sperm. These morphological and biochemical changes confirm that capacitation of L. vannamei sperm takes place following mating.     

日本黄姑鱼精子生理特性及超低温冷冻保存研究

对日本黄姑鱼精子部分生理特性及其超低温冷冻保存技术进行了研究。结果表明,日本黄姑鱼精液pH值为7.0~7.5,精子密度为(11.23±2.78)×109/m l,精子寿命(229.33±17.16)s。在盐度为30~35,pH为7.5~8.5时,精子的活力最高,分别达到(88.33±2.89)%和(88±4.33)%。精子在室温(25℃)条件下,可存活24 h;在低温(4℃)条件下可以存活36 h。以D液作为稀释液,20%的乙二醇作为抗冻剂,利用快速降温法对精子进行超低温冷冻保存,38℃水浴快速解冻,解冻后的精子获得最高的活力为(47±5.78)%。     

Density and Water Exchange-Dependent Growth and Survival of Litopenaeus setiferus Postlarvae

This present study was designed to investigate the effects of stocking density and water exchange on the growth rate, survival and performance index of L. setiferus postlarvae under controlled laboratory conditions. The experiment was done with postlarvae (PL10 to PL40) at densities of 50, 150, 250 and 350 shrimp/m² and various different water exchanges rate per day (0, 6, 12 and 18%). The maximum growth rate was obtained for shrimp with 12% water exchange per day at all densities. A reduction of the maximum growth rate was observed in relation to density with the highest values in shrimp stocked in a density of 50 and 150 shrimp/m² (mean value of 0.53 mg/d) and the lowest in shrimp stocked in a density of 350 shrimp/m² (0.24 mg/d). The multiple regression equation obtained to relate performance index (growth rate* survival : PI), shrimp density (X₁) and water exchange (X₂) was: PI = 0.31 + (0.001) X₁+ 0.039 X₂+ 2.28 × 10⁻⁶ X₁²+ (−0.0017) X₂²+ (0.000026)X₁X₂, R ²= 0.78; P > 0.03. According to this equation the optimum shrimp density-water exchange comhination was between 5 to 12% of water exchange at stocking density of between 50 and 150 shrimp/m². Salinity, ammonia-N and nitrite-N increased according to the time spent in tanks without water exchange. With no (0%) water exchange, water quality parameters measured were outside the optimum for L. setiferus postlarvae. The use of optimum density and water exchange in a nursery system for L. setiferus with optimum variables established is proposed.     

Use of biofloc technology during the pre‐maturation period of Litopenaeus vannamei males: effect of feeds with different protein levels on the spermatophore and sperm quality

The objectives of this study were: (1) Compare two systems for pre‐maturation of Litopenaeus vannamei in terms of spermatophore and sperm quality, (2) Compare the effect of feeds with different protein levels on reproductive quality of males reared in a biofloc‐dominated system. Animals (36.40 ± 3.13 g) reared under biofloc technology (BFT) were used in the 30‐day experiment, which involved four treatments: one in a clear water system (CW) and other three in a BFT system. The BFT treatments were differentiated by feed: mix of fish, squid and crab (BFT+FF) composed of 68.48% dietary protein (DP); broodstock feed (BFT+BF) composed of 52.51% DP; and juvenile feed (BFT+JF) composed of 39.91% DP. Feed in the CW was also the mix of fresh food. Spermatophore and sperm quality were analyzed at the beginning and end of the experiment. Higher normal sperm rate was recorded in the CW compared with the BFT+FF. Among the BFT treatments, the BFT+FF had the lowest normal sperm rate. Thus, the use of BFT for pre‐maturation of L. vannamei allowed the reduction in dietary protein levels from 68.48% (BFT+FF) to 39.91% (BFT+JF) and the maintenance of spermatophore and sperm quality compared to the system based on high daily exchange rate.     

Hollow sperm syndrome during spermatogenesis in the giant tiger shrimp Penaeus monodon (Fabricius 1798) from eastern Australia

During spermatogenesis, giant tiger shrimp (Penaeus monodon) from Queensland, eastern Australia had a high proportion of testicular spermatids that appeared ‘hollow’ because their nuclei were not visible with the haematoxylin and eosin stain. When examined by transmission electron microscopy, the nuclei of hollow spermatids contained highly decondensed chromatin, with large areas missing fibrillar chromatin. Together with hollow spermatids, testicular pale enlarged (PE) spermatids with weakly staining and marginated chromatin were observed. Degenerate‐eosinophilic‐clumped (DEC) spermatids that appeared as aggregated clumps were also present in testes tubules. Among 171 sub‐adult and adult P. monodon examined from several origins, 43% displayed evidence of hollow spermatids in the testes, 33% displayed PE spermatids and 15% displayed DEC spermatids. These abnormal sperm were also found at lower prevalence in the vas deferens and spermatophore. We propose ‘Hollow Sperm Syndrome (HSS)’ to describe this abnormal sperm condition as these morphological aberrations have yet to be described in penaeid shrimp. No specific cause of HSS was confirmed by examining either tank or pond cultured shrimp exposed to various stocking densities, temperatures, salinities, dietary and seasonal factors. Compared with wild broodstock, HSS occurred at higher prevalence and severity among sub‐adults originating from farms, research ponds and tanks. Further studies are required to establish what physiological, hormonal or metabolic processes may cause HSS and whether it compromises the fertility of male P. monodon.     

Electrical stimulation of spermatophore expulsion in marine shrimp,Penaeus spp

Electrical stimulation (4–6 V AC) applied near the male gonopores at the base of the fifth pereopods caused expulsion of the spermatophore in Penaeus setiferus, P. stylirostris, and P. vannamei. For healthy animals, some degree of spermatophore expulsion occurred in 80% of the attempts. Complete expulsion of one or both spermatophores occurred in only 47% (29–65%, depending on species). Shrimp with melanized terminal ampullae generally did not expel a spermatophore following electrical stimulation. The technique needs further refinement for routine use with penaeids.     

克氏原螯虾雄性生殖系统的超微结构

采用透射电镜研究了克氏原螯虾输精管的超微结构,采用石蜡切片法研究了克氏原螯虾的精巢、输精管和精荚的显微结构,并对精巢做了周年变化研究。结果表明,精巢由许多生精小囊和收集管组成,其结构呈葡萄串状,生殖细胞在生精小囊内发育成熟后由收集管输送到输精管;输精管有左右两条,相对于左侧输精管,右侧输精管比其约长一半,直径更大,卷曲程度更高,内容物含量更多,分泌细胞内的内质网含量更丰富;输精管根据形态不同分为前、中、后、生殖突4段,前中段具有分泌精荚壁物质的功能,后段具有储存、射出精荚的功能,末端生殖突为输精管出口;精荚呈长囊形或椭球形,由精子团、精荚基质和两层精荚壁组成,精子团偏向精荚一侧分布;克氏原螯虾雄虾精巢在一周年中的5—8月和10月处于精子细胞期的生精小囊比例很高,具有两个成熟时期。