Development and Deterioration of Spermatophores in Pond-Reared Penaeus vannamei

Spermatophore deterioration in pond-reared Penaeus vannamei was studied, and spermatophore quality was evaluated and improved. Sperm production of males collected from grow-out ponds was increased by two techniques: eyestalk ablation using a fresh frozen maturation diet (13% body weight); and laboratory culture using a diet of the formulated feed Nicovita Plus (3% body weight/ d) and frozen squid (2%). Findings are complementary to previous reports that eyestalk ablation improves quality of spermatophores in young (25.7 g) males. The timing of eyestalk ablation for activation of the endocrine mechanism, leading to improved spermatophore quality was also explored. After eyestalk ablation, performed between postmolt and intermolt stages, 26 g males required a minimum of three spermatophore regenerations or 42 d to significantly increase spermatophore size and sperm count. On the other hand, the laboratory culture (2.5 mo) technique improved the quality of spermatophores in successive regenerations for non-ablated males. In the present study, subadult P. vannamei produced spermatophores which, if not transferred or manually ejaculated, gradually deteriorated (successive stages are described), while a new compound spermatophore was being synthesized.     

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.     

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.     

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.     

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.     

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 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.     

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.     

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 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.     

Dietary Effects on Sperm Quality of Litopenaeus vannamei Broodstock

A 56‐d feeding trial was conducted to investigate the effect of diet on sperm quality of Pacific white shrimp Litopenaeus vannamei broodstock. Dietary treatments consisted of a combination of 75% dry maturation diet and 25% fresh‐frozen squid (dry‐weight basis). Supplemental nutrients of the maturation diet were selectively deleted and replaced with wheat starch to produce the following treatments: 1) 75% basal maturation diet plus 25% squid (control); 2) 75% maturation diet without supplemental vitamins plus 25% squid; 3) 75% maturation diet without supplemental cholesterol and phospholipids plus 25% squid; 4) 75% maturation diet without supplemental astaxanthin plus 25% squid; and 5) a fresh diet composed of 60% squid and 40% Maine bloodworms. Shrimp fed the control diet and the diet without supplemental astaxanthin had significantly higher mean (± SEM) change in sperm count (4.6 ± 3.2 million sperm cells and 2.9 ± 2.5 million sperm cells, respectively), with respect to baseline (8.7 ± 1.0, 6.4 ± 1.0, 9.0 ± 1.3, 6.6 ± 0.7, and 6.0 ± 0.8 million sperm cells for treatments 1, 2, 3, 4, and 5, respectively), than shrimp fed the diet without supplemental vitamins (‐1.7 ± 2.6 million sperm cells), but not significantly higher than those of shrimp receiving the diets without supplemental cholesterol‐phospholipids (1.2 ± 2.5 million sperm cells) and the fresh diet (1.3 ± 1.6 million sperm cells). Dietary deficiencies also were reflected in weight gain of shrimp fed the diet without supplemental vitamins (‐2.0 g) and the fresh diet (‐0.8 g). which were significantly lower than weight gain of shrimp fed the control diet (1.1 g) and the diet without supplemental cholesterol‐phospholipids (0.8 g). No significant differences were detected among treatments for percentage of abnormal sperm and survival data. Results demonstrated a significant effect of diet on reproductive quality of male L. vannamei and indicated that the typical combination of fresh‐food organisms used is not nutritionally optimal for male broodstock.     

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.     

A technique for electrically stimulating extrusion of spermatophores from the lobster, Homarus americanus

A technique for electrically inducing spermatophore extrusion from live lobsters (Homarus americanus) is described. When a 12-V stimulus is applied around the coxa of the fifth walking leg, a single spermatophore is extruded through the gonopore. The sperm in extruded spermatophores are morphologically normal and undergo normal acrosome reactions. The technique provides a ready source of viable sperm for in vitro studies on fertilization and for artificial insemination of freshly molted female lobsters. The spermatophore itself is a natural package for sperm storage and may be useful in the future development of lobster sperm banks.     

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.     

Sperm vitrification of <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>: effect of cryoprotectant solutions on sperm viability and spawning quality after artificial insemination

Vitrification is a fast freezing method with promising results for penaeids sperm cryopreservation. This study evaluated the efficiency of three cryoprotectant solutions for sperm vitrification and artificial insemination with cryopreserved spermatophores of Litopenaeus vannamei. The cryoprotectant solutions tested were 30% methanol, 2% soy lecithin, and 30% methanol?+?2% soy lecithin. Fully mature females were artificially inseminated with vitrified and fresh spermatophores as a control group. The vitrification method was efficient in maintaining high rates of sperm survival and membrane integrity. Although the egg fertilization was successfully attained by artificial insemination with cryopreserved spermatophores, low hatching rates suggested that possible DNA fragmentation of sperm cells should be further investigated. This is the first report of artificial insemination using vitrified sperm in penaeids.     

Evaluating the impact of moulting and chilled storage of spermatophores on the integrity of plasma membrane,acrosome and DNA of black tiger prawn (Penaeus monodon) spermatozoa

To explore the impact of moulting and short‐term chilled storage of spermatophores on the sperm quality for a commercially important penaeid prawn, spermatozoa of Penaeus monodon from early (B‐C), middle (D0‐1) and late (D2‐3) moult stages were compared for sperm quality parameters relating to the structural integrity of plasma membrane (Viab%), acrosome (AR%) and DNA (SDF%) after being stored at 4°C for 0–26 days. The three different sperm extenders used for chilled storage included artificial lobster haemolymph (AH), calcium free saline (CS) and filtered seawater (FS); two storage conditions were applied either as a free sperm suspension or retained within the intact spermatophore. Results showed that (a) the lowest natural AR% was shown for B‐C spermatozoa in CS whereas the highest levels were for B‐C, D0‐1 and D2‐3 spermatozoa in FS and D2‐3 spermatozoa in AH; (b) the calcium ionophore A23187 agent used in this study was able to increase the mean AR% by 6.22%; (c) the Viab% was significantly lower in CS than that in FS; (d) the SDF% significantly increased over the period of chilled storage for B‐C and D0‐1 spermatozoa, while the SDF% of D2‐3 spermatozoa was initially elevated and did not change significantly over time; and (e) there was no difference in sperm quality between two storage conditions. This study has successfully demonstrated the moult‐related variance in the percentages of acrosome‐reacted and DNA‐damaged spermatozoa, providing evidence of moult‐related spermatophore renewal cycle in this species.     

Ammonia-N efflux rate and nutritional state of juvenile pink shrimp, Penaeus paulensis (Perez-Farfante), in relation to food type

The effects of squid flesh, two pelleted diets containing 41% and 53% protein, and starvation on the ammonia-N efflux rate and the biochemical composition of pink shrimp, Penaeus paulensis (Perez-Farfante), were investigated. A transient increase in the ammonia-N efflux rates of shrimp subjected to starvation was observed. Ammonia-N efflux rates of shrimp fed the diet with 41% protein were lower than in those fed the diet with 53% protein or squid flesh. Ammonia-N efflux rates were inversely related to body weight, except during starvation, which affected mainly small individuals. Increased ammonia-N excretion rates increased immediately after the ingestion of high-protein diets. Such rates were also found to be higher during light periods (cf. dark periods), possibly reflecting differences in activity levels. Dietary-related alterations in the blood, muscle and midgut gland constituents were also detected. In general terms, starvation tended to reduce the levels of such constituents, whilst pelleted diets promoted increases in lipid and carbohydrate concentrations.     

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.     

Squid Protein Effect on Growth of Four Penaeid Shrimp

The growth response to supplementation of mixed diets with a protein extracted from frozen squid (squid protein fraction: SPF) was tested at levels of 1.5, 3.0, 6.0 and 16.0% in the diet in four species of shrimp: Penaeus stylirostris, P. vannamei, P. monodon and P. indicus. In P. stylirostris and P. vannamei , growth rates were significantly improved by SPF even at the lowest level of supplementation. Improvement was obtained only with 6 and 16 percent in P. monodon , while no significant response was observed in P. indicus. The growth promoting effect of SPF at 16% of the diet could be explained by its nutritional value, particularly by its amino acid content, although all diets were supposed to be well balanced in all known nutrients. However, at lower levels the results obtained seemed to be due to the unknown growth factor (UGF) already mentioned in previous reports.     

Chilled storage of banana shrimp (Fenneropenaeus merguiensis) spermatophores with supplementation of moringa (Moringa oleifera Lam.) extract

This study was designed to optimize the chilled storage method for banana shrimp (Fenneropenaeus merguiensis) spermatophores and evaluate the potential of moringa (Moringa oleifera Lam.) extract on the reduction in bacterial contaminants during spermatophore preservation due to the uncertainty of the broodstock. Spermatophores were suspended in five extenders: mineral oil, Ringer's solution, phosphate buffer, calcium‐free saline and 0.8% NaCl and stored at 2–4°C. During a 28‐day storage, spermatophores stored in mineral oil showed the highest sperm viability (89.1%) and intact morphology with a slight formation of hardened adhesive matrices. The effect of moringa extract was investigated on chilled spermatophores. Spermatophores were suspended in mineral oil (the control) and mineral oil containing either penicillin–streptomycin (0.1%) or moringa extract (0.1 mg/ml) during a 28‐day storage. Supplementation of moringa extract resulted in a significant increase (p < .05) in sperm survival, compared to the control, and a complete elimination of culturable Vibrio (Vibrio alginolyticus, Vibrio mimicus and Vibrio furnissii), Staphylococcus kloosii, Bacillus macerans, Listeria ivanovii, Corynebacterium paurometabolum and Corynebacterium bovis, in chilled spermatophores. Chilled storage of spermatophores in mineral oil containing moringa extract was a promising technique due to the inhibition of shrimp and human pathogens without the spermicidal effect on banana shrimp sperm.