Histological examination of final oocyte maturation and atresia in wild and domesticated Penaeus monodon (Fabricius) broodstock

Oocyte maturation and gonadosomatic index (GSI) of eyestalk ablated Penaeus monodon females collected from the wild and from two first‐generation domesticated lines were assessed. Frequency and diameter of the different oocytes, and the intensity of oocyte atresia, were compared among groups through histological assessments of the sections of the middle ovarian lobe. Digitized images from ovary sections were used to record the frequency and diameter of different oocyte types. Spawning performance of the three groups were expressed in terms of the percentage of females that spawned at least once (productive females), time from eyestalk ablation to first spawning (latency period) and the number of spawnings per female stocked. Final ovarian maturation was attained in all groups, as indicated by the presence of mature oocytes with cortical rods (cortical oocytes), dark‐green ovarian colour and high GSI values (5.83–6.86%). However, domesticated groups presented significant larger immature oocyte types (previtellogenic and yolky oocytes) and smaller cortical oocytes compared with wild females, indicating a reduced vitellogenic activity during final oocyte maturation. Additionally, the frequency of atresia was comparatively higher for both domesticated groups, which could be related to their inferior spawning performance. The implications of these results on the reproductive potential and development of domesticated P. monodon are discussed.     

中国对虾卵子激活过程的形态学研究

报道了对中国对虾卵子激活反应的形态学研究。中国对虾卵子皮层中有由卵黄膜下陷形成的与卵表垂直的皮层囊,内有棒状结构称棒状周边体,其内物质为凝胶前体。对虾卵子的激活并不需要精子的发动,而是在卵子与海水接触后即开始进行。在卵子激活过程中,棒状周边体向外放出形成花冠状的凝胶层,然后从基部开始逐渐消失并向远端扩展,最后完全消失,同时卵子进行成熟分裂放出第一、第二极体,并举起受精膜,完成激活反应。还对中国对虾卵子激活的作用进行了讨论。     

Biochemical characterization and physiological role of cortical rods in black tiger shrimp, Penaeus monodon

Cortical rods (CRs), precursors of egg jelly investment in many penaeoid shrimp, are composed of different proportions of proteins and carbohydrates, the physiological role of which still requires extensive investigation. In this study, we demonstrated the biochemical properties of the CRs and their role in the induction of the acrosome reaction (AR). Profiles of the isolated CRs revealed a number of major protein bands ranging from 35 to 230 kDa. These CR proteins were extensively glycosylated and sulfated. Lectin-based carbohydrate analysis further revealed the highest reactivity of concanavalin A (Con A) among other lectins used. In addition, the selective interference of Con A binding with mannose but not glucose indicated that CR glycoproteins were of high-mannose type. Using immunoblotting with anti-CR antibody, we further demonstrated that part of egg water (EW, a natural AR inducer) was derived from miscible components of the CRs. Physiological tests of water-soluble CR (wsCR) revealed its high AR inducing competency comparable to that of EW, which was far superior to that of acid-urea treated CR (auCR). Furthermore, the wsCR-induced AR was selectively inhibited by Con A, suggesting the significance of the exposing mannose residues in regulating P. monodon sperm AR response.     

Spawning of female Black tiger shrimp (Penaeus monodon) is not impacted by muscle injection of dsRNA targeted to gill‐associated virus

The ability of domesticated Penaeus monodon, Black Tiger shrimp, to spawn following tail‐muscle injection of dsRNA was examined. Ablated domesticated female broodstock infected subclinically with gill‐associated virus (GAV) were injected with saline or a cocktail of five‐dsRNAs targeting different regions in the GAV ORF1a/1b gene. To track changes in GAV infection loads, TaqMan real‐time PCR was used to quantify mean viral RNA amounts in each of three pleopod clips collected at the time of injection (Day 0) and either immediately after a female spawned or on Day 11 when the trial was terminated. Over the trial, 4 of 19 (21%) saline‐injected shrimp spawned and 12 of 25 (48%) dsRNA‐injected shrimp spawned, with one spawning twice. Egg numbers varied from 25 600 to 459 800 for the saline‐injected shrimp and from 4900 to 213 900 for the dsRNA‐injected shrimp. Of these, one of the four egg batches hatched from saline‐injected shrimp and 9 of the 13 egg batches hatched from dsRNA‐injected shrimp. While variable, egg numbers and hatch rates recorded were typical of those obtained from domesticated broodstock at the commercial hatchery and particularly among females previously spawned. Mean GAV RNA amounts detected in pleopod samples increased in five of the eight saline‐injected shrimp tested by 1.6–227.4‐fold and decreased in 12 of the 15 ds‐RNA‐injected shrimp tested by ?1.1 to ?45.1‐fold. The study demonstrated that tail‐muscle injection of GAV‐specific dsRNA does not adversely impact the ability of P. monodon to spawn.     

中国对虾精子入卵过程的观察

利用光镜和电镜对中国对虾精子入卵过程进行了观察。结果表明，精子的核物质籍胞吐作用穿过卵黄膜到达卵膜，其余部分则留在卵黄膜外。只有在卵子激活前到达卵膜的精子才能参与受精作用，其余的精子将被卵子激活所形成的胶质层推离卵子。中国对虾精子激活过程，没有发现顶体丝的形成。精子的入卵开始于卵子的皮层反应后期，此时精卵的接触逐渐密切，首先接触部位的卵膜稍有突起，然后开始下陷，同时这一部位的卵膜变得粗糙不平，随后卵膜解体，精子由此进入卵子。中国对虾精子的入卵位置是随机的，有多精入卵的现象。     

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.     

锯缘青蟹精子入卵过程的扫描电镜观察

利用扫描电镜详细观察了锯缘青蟹精子入卵的过程。精子以其核突起附着在卵膜上,并迅速发生顶体反应。顶体反应时,顶体囊外翻,顶体管前伸,精子核辐射臂收缩,并拖至顶体囊的后部。顶体管迅速穿过卵黄膜,携带核物质一同进入卵子。锯缘青蟹为多精着卵,数精入卵。本文同时探讨了精子顶体反应机制以及受精过程卵子的作用。     

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.     

Effect of arachidonic acid supplementation on reproductive performance of tank‐domesticated Penaeus monodon

The reproductive performance of domesticated Penaeus monodon was assessed when fed on two experimental semi‐moist maturation diets varying in their arachidonic acid content for 21 days before ablation and throughout a 17‐day reproductive assessment. The biochemical composition of the two semi‐moist two diets was similar with the exception of arachidonic acid (ARA) content; the basal diet (BAS) consisting of 0.9 g kg^?1 DM ARA (1.1% of total fatty acids) and the supplemented diet (ARA‐SUP) consisting of 5.0 g kg^?1 DM ARA (5.8% of total fatty acids). ARA/EPA and ARA/DPA ratios were 0.1 in the BAS diet and 0.5 in the ARA‐SUP diet. Fatty acid composition of the spawned eggs was comparable between diets with the exception of ARA concentration, which was higher in the ARA‐SUP (8.95 ± 0.44 g kg^?1 DM) than the BAS (3.23 ± 0.17 g kg^?1 DM) (P < 0.0001). The cumulative percentage of females spawning (mean ± SE after 17 days) (31.9 ± 7.0%; 24.1 ± 1.3%), number of spawnings per female (0.48 ± 0.1; 0.29 ± 0.02), and eggs per female (62 520 ± 16 935; 44 521 ± 9914) was significantly (P < 0.0001) higher for the ARA‐SUP than the BAS. Results of this study suggest that arachidonic acid plays a key role in promoting egg development and spawning in P. monodon.     

Serotonin stimulates ovarian maturation and spawning in the black tiger shrimp Penaeus monodon

Serotonin (5-hydroxytryptamine, 5HT) has been reported to induce ovarian maturation and spawning in the crayfish Procambarus clarkii and white Pacific shrimp Litopenaeus vannamei. The aim of this study was to explore the role of exogenous 5HT on the reproductive performance of the black tiger shrimp Penaeus monodon. 5HT solution was injected into domesticated P. monodon broodstock at 50 μg/g body weight and ovarian maturation and spawning were recorded. The presence of 5HT in the ovary and oviduct of P. monodon was also studied by immunohistochemistry and its levels in the ovary by enzyme link immunoabsorbance assay (ELISA). The 5HT-injected P. monodon developed ovarian maturation and spawning rate at the level comparable to that of unilateral eyestalk-ablated shrimp. Hatching rate and the amount of nauplii produced per spawner were also significantly higher in the 5HT-injected shrimp, compared to the eyestalk-ablated shrimp. 5HT-positive reactions were found in the follicular cells of pre-vitellogenic oocytes, in the cytoplasm of early vitellogenic oocytes and on the cell membrane and cytoplasm of late vitellogenic oocytes. 5HT in the ovary was present at 3.53 ± 0.26 ng/mg protein level in previtellogenic stage and increased to 17.03 ± 0.57 ng/mg protein level in the mature stage of the ovary. The results suggest a significant role of 5HT, possibly directly on the ovary and oviduct, on the reproductive function of female P. monodon.     

Environmental characteristics of spawning and nursery grounds of Japanese sardine and mackerels in the Kuroshio and Kuroshio Extension area

The sustainable use of marine resources requires understanding the surrounding ecosystem and elucidating mechanisms of variation. However, we still lack a comprehensive understanding of environmental variation in the spawning and nursery grounds of important fisheries species Japanese sardine (Sardinops melanostictus) and mackerels (Scomber japonicus and Scomber australasicus) in the northwest Pacific. Here, we investigate detailed physical, chemical, and biological environment variations in the spawning and nursery grounds along the Kuroshio and Kuroshio Extension area from intensive investigation in spawning season (April) of 2013. We found similar water mass property and copepod community in the egg‐rich Kuroshio area and the larvae‐rich downstream Kuroshio Extension area, indicating environmental variability is small during transportation and development processes. The egg‐rich northern Izu Islands region showed high copepod abundance, although low nutrient and chlorophyll concentrations were observed. Eggs were scarce or absent in the second survey 10 days after abundant eggs were observed in the region, along with differences in water property and copepod community. This indicates that not only the location but also the specific water characteristic and copepod community are a determining factor for spawning. Indicator communities of copepod found in our study (indicator community of transportation process from spawning ground, of non‐spawning ground, and of reproductive area in the Kuroshio Extension area) would be a key factor for recruitment prediction.     

Ultrastructural changes in Marsupenaeus japonicus spermatozoa in the journey from the testis to the female thelycum

Marsupenaeus japonicus is one of the most important penaeid shrimp farmed in China. As a typical closed thelycum penaeid shrimp, the morphological changes in the spermatozoa during the journey from the testis to the female thelycum remain unclear in this species. Herein, an ultrastructural comparison of the spermatozoa in the testis (TE), vas deferens (VD), and the spermatophore in the male terminal ampoule (SA) and the female seminal receptacle (SR) of M. japonicus was conducted. The spermatozoa in the VD, SA and SR shared typical morphological characteristics of the spermatozoa of decapod dendrobranchiates, including a spherical main body and a slender spike. However, no spike was observed in the spermatozoa collected from the TE. The spike of the spermatozoa in the SA and SR was longer than that in the VD, whereas no significant difference in spike length was observed between the spermatozoa from the SA and SR. The basic ultrastructure of the spermatozoa in the VD, SA and SR was similar, except for some differences in the nuclear region. The main body of the spermatozoa comprised the cytoplasmic band, nuclear region, several small and large vesicles, and the basal region of the acrosomal structure. The nuclear materials in the spermatozoa from the TE were more electron‐dense than those in the VD, SA and SR, while the nuclear region of the spermatozoa was more electron‐lucent in the SR than in the VD and SA. These findings preliminarily reveal the ultrastructural changes in M. japonicus spermatozoa during the journey from the testis to the female thelycum and provided a theoretical foundation for developing effective artificial insemination methods in this species.     

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     

Alternating dominance of postlarval sardine and anchovy caught by coastal fishery in relation to the Kuroshio meander in the Enshu-nada Sea

In the mid 1970s, the fishery catch of postlarval Japanese anchovy (Engraulis japonica) in a shelf region of the Enshu‐nada Sea, off the central Pacific coast of Japan, started to decline corresponding to a rapid increase of postlarval sardine (Sardinops melanostictus). In late 1980s, sardine started to decline, and it was replaced by anchovy in the 1990s. This alternating dominance of postlarval sardine and anchovy corresponded to the alternation in egg abundance of these two species in the spawning habitat of this sea. It was also noteworthy that during the period of sardine decline, sardine spawning occurred in April–May, a delay of two months compared with spawning in the late 1970s. The implication of oceanographic changes in the spawning habitat for the alternating dominance of sardine and anchovy eggs was explored using time‐series data obtained in 1975–1998, focusing on the effect of the Kuroshio meander. Large meanders of the Kuroshio may have enhanced the onshore intrusion of the warm water into the shelf region and contributed to an increase in temperature in the spawning habitat. This might favour sardine, because its egg abundance in the shelf region was more dependent on the temperature in early spring than was that of anchovy. In addition, enhanced onshore intrusion could contribute to transport of sardine larvae from upstream spawning grounds of the Kuroshio region. On the other hand, anchovy egg abundance was more closely related to lower transparency at the shelf edge, which may indicate the prevalence and prolonged residence of the coastal water, and therefore higher food availability, frequently accompanying non‐meandering Kuroshio. The expansion/shrinkage of the spawning habitat of sardine and anchovy in the shelf region, apparently responding to the change in the Kuroshio, possibly makes the alternation in dominance of postlarval sardine and anchovy most prominent in the Enshu‐nada Sea, in combination with changes in the abundance of spawning adults, which occurred almost simultaneously in the overall Kuroshio region. The implication of this rather regional feature for the alternating dominance of sardine and anchovy populations on a larger spatial scale is also discussed.     

Acrosome reaction of Chinese mitten-handed crab Eriocheir sinensis (Crustacea: Decapoda) spermatozoa: Promoted by long-term cryopreservation

The effects of three media, two temperatures, and fourteen durations of cryopreservation from 0 h to 450 d on in vitro acrosome reaction (AR) of spermatozoa in Chinese mitten-handed crab Eriocheir sinensis (Crustacea: Decapoda) were investigated. The spermatozoa of good quality were obtained from spermatophores by a glass homogenizer in an ice-bath and centrifugation at 4 °C. At 0 h, 2 h, 1 d, 3 d, 15 d, 30 d, 60 d, 90 d, 120 d, 150 d, 180 d, 270 d, 360 d, and 450 d of cryo-storage in Ca²⁺-free artificial seawater, 10% (v/v) glycerol, and 5% (v/v) dimethylsulfoxide at − 80 °C and in liquid nitrogen (− 196 °C), the changes in spermatozoal morphology, the time of beginning AR, and the time of maximum percentage of AR were observed. The relationships of the changes on AR presented with the different media, temperatures, and durations of cryopreservation were speculated. In this study, the cryopreserved spermatozoa all underwent AR in less than 1 h of settlement under room temperature while the percentage of AR in the control was only about 4.9%. Meanwhile, cryopreservation shortened both the time of beginning AR (from 30.11 min of the uncryopreserved spermatozoa to 0 min of cryopreserved spermatozoa on the 30th day) and the time of maximum percentage (from 59.88 min of the uncryopreserved spermatozoa to 0 min of cryopreserved spermatozoa on the 60th day). Whereas the effect of media on sperm cell AR was negligible (P > 0.05), the treatments of spermatozoa with short- and long-term cryopreservation resulted in extremely significant differences in the time of beginning AR as well as in the time of maximum percentage of AR (P < 0.01). The present data indicate that cryopreservation for long or short periods can promote the AR of sperm cells in E. sinensis and physiologically affect the ability to capacitate. It may be that the mechanism of AR in this study is the direct promotion of membrane fusion of the acrosomal cap, or destruction of the proteins inhibiting AR and activation of the proteins promoting AR, by cryopreservation. In addition, the results also show that cryopreservation can protect the spermatozoa because AR can occur in almost all sperm cells cryopreserved for less than 15 d.     

Effects of different feeding regimes on ovarian maturation and spawning of pond-reared giant tiger prawn in Thailand

Large pond-reared Penaeus monodon were used in three maturation and spawning trials lasting 60 days. One group of prawns was fed a fresh diet only; a second group was supplied a fresh diet combined with a formulated pelleted diet; a third group of prawns received the formulated diet only. Total maturation (N = 66 and 55) and spawning events (N = 63 and 48) were much greater for prawns receiving only the fresh diet, and the combined fresh with formulated diets, respectively. Prawns receiving only the formulated diet matured only 12 times, and spawned 11 times. Prawns fed a fresh diet produced significantly more eggs than those given a formulated diet alone. No significant differences were seen in eggs produced per spawning event, % fertilization, % hatch or % metamorphosis from egg to protozoea. Female prawns exhibited greater mortality than did males, while females on the fresh diet only had greater mortality than did other females, presumably due to greater handling and other stress factors. Female prawns on a combined diet had lower mortality than other females. The formulated diet used is suitable for supplementation, but not as a sole maturation food.     

Productivity benefits of selectively breeding Black Tiger shrimp (Penaeus monodon) in Australia

Advances in the domestication and selective breeding of Australian Black Tiger shrimp, Penaeus monodon, opens the opportunity for world producers to reconsider the benefits of farming this species. Just over a decade ago this species was the world's most farmed shrimp species, however, difficulty in its domestication, in part, led to the widespread establishment of Penaeus vannamei (Pacific White shrimp) as the most farmed shrimp species in the world. This study empirically evaluates the productivity benefits of commercially domesticating P. monodon against production from wild broodstock of the same species. The evaluation compared the relative production from commercial ponds stocked with the progeny of wild P. monodon broodstock and ponds stocked with the progeny of domesticated stocks. The production data were from 164 ponds of domesticated stocks and 30 ponds of wild stocks, collected over 4 years (2009–2013) from two separate farm sites of the same Australian shrimp farming company. The wild stocks were sourced from the east coast of Australia. The results suggested that the productivity of the selectively bred stocks was 39% greater compared with production from wild stocks given equivalent amounts of feed and other inputs. Furthermore, productivity was additionally enhanced depending on the choice of feeds and whether stocking took place in September rather than later in the year (i.e. in early spring rather than late spring/early summer in the Southern hemisphere). This suggests that there is significant potential to further enhance the productivity of P. monodon farms via integrating advances in domestication, feeds and management practices.     

Tolerance of Penaeus monodon Fabricius embryos to ozonated seawater

The tolerance of Penaeus monodon embryos from five spawnings (families) to four different ozone doses in seawater [0.0, 0.5, 1.0 and 2.0 mg L⁻¹, measured by the residual oxidant concentration (ROC)] was examined when applied for three exposure times (1, 2 and 4 min) at three post‐spawning treatment times (25, 120 and 480 min post‐spawning). Ozone dose typically had a larger affect on embryo hatching than exposure time and the ozone dose × exposure time interaction for most combinations of family and post‐spawning treatment time. At ozone doses of 2.0 mg L⁻¹, embryos had lower hatchings than controls for all families at 25 and 120 min post‐spawning, and for several combinations of family and exposure time at 480 min post‐spawning. At ozone doses of 1.0 mg L⁻¹, the effect on embryo hatching was more varied between families and exposure times for the three post‐spawning treatment times, but typically embryos were less affected when exposed at later post‐spawning treatment times. Ozone doses of 0.5 mg L⁻¹ typically had minimal effects on hatching for all exposure times and post‐spawning treatment times. In summary, later‐stage P. monodon embryos typically tolerated ozone doses of up to 1.0 mg L⁻¹ in seawater for durations of up to 4 min.     

Several possible spawning sites of the Japanese eel determined from collections of their eggs and preleptocephali

The spawning area of the Japanese eel is located at the southern part of the West Mariana Ridge in the western North Pacific, but their spawning events have not been observed. To further understand Japanese eel spawning ecology, an interdisciplinary research survey by the R/V NATSUSHIMA (NT14-09, 14 May–4 June 2014) was conducted to detect spawning sites based on the seamount, salinity front, new moon and third quadrant (spawning south of front, west of ridge) hypotheses. Attempts were made to film spawning events with underwater camera systems and to consider if eels might be detected in hydroacoustic observations. Although no Japanese eels or spawning events were video-recorded and no eel aggregations could be clearly identified acoustically, three eggs were collected at two stations in the third quadrant region at or just south of 13° N on 26 and 27 May. Three or four days later, newly hatched preleptocephali were collected at two stations far to the south, including 224 at a station > 160 km southwest of the egg catches, and a few preleptocephali were caught at two stations closer to the egg stations. The eggs and southern preleptocephali were from discrete spawning events, which indicated that at least two spawning sites occurred in May 2014.

     

Evaluation of Commercial‐scale Approaches for Cryopreservation of White Crappie,Pomoxis annularis,Sperm

Crappie, Pomoxis spp., are popular game fish throughout North America and are produced by public and private hatcheries. However, production is limited by a lack of information on tank culture and induced spawning methods. Development of techniques for storage of sperm and in vitro fertilization would increase flexibility in spawning. Therefore, techniques for sperm cryopreservation were examined in white crappie, Pomoxis annularis. Sperm from adult wild white crappie were used to evaluate sperm extender, cryoprotectant agent and concentration, and cooling technique based on post‐thaw sperm motility. Percent egg fertilization was also compared between sperm stored in the two best cryopreservation protocols and two different osmotic activator solutions. Sperm were cryopreserved using treatment combinations of two extenders (350 mOsmol/kg Hanks' balanced salt solution [HBSS] and 350 mOsmol/kg Ca²⁺free HBSS) and two cryoprotectants (dimethyl sulfoxide [DMSO] and methanol) at concentrations of 5, 10, and 15% that were cooled at four different rates: 5, 10, 20, and 40 C/min. Post‐thaw sperm motility and fertilization rates indicated white crappie sperm can be cryopreserved using either extender, cryoprotectants of either 5% DMSO or 10% methanol, and cooling at 40 C/min. A follow‐up experiment demonstrated sperm in suspensions on ice retained viability after overnight transport.