Sperm cryopreservation in guppies and black mollies—A generalized freezing protocol for livebearers in Poeciliidae

doi:10.1016/j.cryobiol.2009.09.011

Cryobiology

Volume 59, Issue 3, December 2009, Pages 351-356

https://doi.org/10.1016/j.cryobiol.2009.09.011 Get rights and content

Abstract

In this study, we evaluated various parameters of sperm cryopreservation in two livebearers, guppies (Poecilia reticulata) and black mollies (P. latipinna). Our results suggested a common freezing protocol for the guppies and mollies: suspend sperm in Hanks’ balanced salt solution (HBSS) at 300 mOsm/kg, use 14% glycerol as cryoprotectant, cool at 25 °C/min, and thaw at 40 °C in a water bath for 7 s. Live young were produced from females inseminated with frozen-thawed sperm in both species. In guppies, percent fertilization (F) and the number of embryos (N) produced with cryopreserved sperm (F = 50%, N = 74, from 26 females) were similar to those of fresh controls (F = 54%, N = 61, from 22 females). Interestingly, this same freezing protocol has been used successfully for sperm cryopreservation in green swordtails Xiphophorus helleri, and platyfish of X. couchianus with post-thaw motility as high as 80%. All these species belong to the family of Poeciliidae, and their sperm are similar in morphology exhibiting the absence of acrosome, elongate sperm head, and the long mitochondrial sheaths. Besides their internal fertilization reproduction mode, these fish are also small in size (2–4 cm) and live in a freshwater environment. Sperm cryopreservation in fish has been generally recognized as species specific, and new protocols are required for new species. However, results presented in this study suggested otherwise. Thus, sperm cryopreservation methods optimized for one species may be applicable to others if they are taxonomical closely related species with similar sperm morphology and reproduction mode. Considering the enormous number of fish species on the planet, development of generalized sperm freezing protocols for species in groups could have additional advantages for genetic conservation.

Introduction

In comparison with mammalians, sperm cryopreservation protocols in fish generally are more heterogeneous. For example, TES–tris solution supplemented with egg yolk, glucose, and glycerol is a common recipe for sperm cryopreservation in various mammals, however, a wide range of cryoprotectants have been reported for various fish species [3]. One possible explanation for such divergent responses of fish sperm to cryopreservation is because fish are diverse in sperm morphology, reproduction modes, and habitats [12]. Another possible reason is that sperm cryopreservation in fish in general is less well characterized for any given species and thus more unified freezing protocols are difficult to derive for species with similar reproductive characteristics. In fact, sperm cryopreservation in fish has been generally recognized as species specific, and new protocols are required for new species. Still, many fish species in the same taxonomical group (e.g., genus or family) live in the same habitats (e.g., freshwater, blackish water, and salt water), have similar sperm morphology (head, middle piece, and flagellum), and share the same reproduction mode (external vs. internal). Thus sperm from the same group of fish are more likely to have similar biophysical properties in response to osmotic shock and freezing or cooling processes. Therefore, it is possible that species in the same group could utilize a common freezing protocol.

Guppies (Poecilia reticulata) and mollies (P. latipinna) of the genus Poecilia along with swordtails and platyfish of the genus Xiphophorus are viviparous teleosts of the family Poeciliidae. They are also known as the “big four” within aquarium trade as the most popular ornamental species [25]. In addition, guppies and members of Xiphophorus are important experimental model fish for diverse fields of contemporary scientific research (e.g., [21], [28]). As viviparous species, they represent an atypical reproductive mode of internal fertilization in teleost fishes, which are usually characterized as oviparity with external fertilization. Similarly, their sperm are also different from that of fishes that employ external fertilization. Sperm of livebearers in Poeciliidae have elongated sperm heads [12], well-developed mitochondrial sheaths in the midpiece [22] (Fig. 1), and glycolytic activity comparable to mammalian species [7]. In addition, these sperm can retain continuous motility for days as adaptations for movement or long-term survival within the female reproductive tract after copulation [9]. After release into the female genital tract, these sperm can also be stored alive for months before fertilization of mature oocytes [20], and females may produce 4–5 broods after a single insemination [26].

Sperm cryopreservation in fish has been mainly focused on species with external fertilization, and studies on species with internal fertilization have just been initiated recently [9], [10], [11]. Sperm cryopreservation in guppies and mollies has not been explored. In this study, we evaluated various parameters of sperm cryopreservation in guppies and black mollies, and compared with our previous findings on other livebearers in the same family such as the green swordtails Xiphophorus helleri [10] and platyfish X. couchianus [11].

Section snippets

Sperm collection and processing

Sexually mature male guppy and molly fish were purchased from the pet market of Wenzhou, and held in a 20 L glass tank at 25 °C on a 14:10 dark/light photoperiod for 2 weeks prior to experiment. The fish were anesthetized in 0.01% tricaine–methane sulfonate for 2 min, and their standard lengths (tip of snout to the crease of the caudal peduncle) and wet body masses were measured. Testes were collected by the methods described previously [9], and were placed in resealable plastic bags and weighed

Effect of cryoprotectant toxicity before freezing

Guppy sperm motility decreased with increasing cryoprotectant concentration except for glycerol, and with increase in storage time (Fig. 2). At 20 min after refrigerated storage, high motility was observed for 5–20% glycerol (35–45%), 5–10% DMSO (37–45%), 5% methanol (40 ± 5%), and followed by 5% DMF (25 ± 8.7%). At 2 h after storage, high motility was only observed in treatments with glycerol and 5% DMSO. Throughout the experiment, sperm motility in the samples with 5%, 10%, and 15% glycerol were

Discussion

This study revealed a common freezing protocol for the guppies and mollies: suspend sperm in HBSS at 300 mOsm/kg, use 14% glycerol as cryoprotectant, cool at 25 °C/min, and thaw at 40 °C in a water bath for 7 s. Live young were produced from females inseminated with frozen-thawed sperm in both species. Previous studies on green swordtails X. helleri [10] and platyfish X. couchianus [11] revealed similar freezing protocols. As indicated earlier, all these four species employ internal fertilization

Conflict of interest

No conflict of interest interfered with the interpretation of the results and that of their applications.

Acknowledgment

We thank S. Rodenburg for English editing.

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To date, little attention has been paid to identifying the effects of long-term cryopreservation on sperm quality for Piaractus orinoquensis. The object of this study was therefore to evaluate the effect of long-term cryopreservation (24 h, 1, 6 and 12 months) on sperm motility, viability, DNA integrity, ATP content, total antioxidant capacity (TAC), morphology and sperm ultrastructure in this species. Milt samples from six males were cryopreserved in a medium containing final concentrations of 7.5% Me₂SO, 4.1% glucose and 9.0% egg yolk. The samples were frozen in liquid nitrogen (LN) vapor and stored in LN for periods of 24 h and 1, 6 and 12 months. After thawing, both the motility rate and the viability decreased significantly compared with fresh sperm; however, these parameters did not differ among the four cryopreservation times. The DNA integrity and ATP content decreased significantly after 6 months of cryopreservation. There were no significant differences in TAC values between fresh and cryopreserved sperm. The total sperm abnormalities in cryopreserved samples were about 5-fold higher than in fresh sperm; short tail was the most common defect occurring after cryostorage. The ultrastructural analysis reveals that P. orinoquensis spermatozoa consist of an ovoid head without acrosome, a cylindrical mid-piece, and a single flagellum. The nuclear fossa is located at the base of the nucleus and contains the centriolar complex. There are 1–2 ring-shaped mitochondria located in the mid-piece. The flagellum shows a 9 + 2 organization of microtubules in the axoneme. Post-thaw spermatozoa presented damage such as swelling and rupture of the plasma membrane, mitochondrial damage, loss of the electron-dense chromatin of the nucleus, and degeneration in the middle region.
Cryopreservation of coho salmon sperm (Oncorhynchus kisutch): Effect on sperm function, oxidative stress and fertilizing capacity
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This is one of the first studies of coho salmon sperm cryopreservation and the first time that the effect on sperm functional parameters has been assessed using CASA and flow cytometry. Generally, the development of fish sperm cryopreservation protocols is species-specific, or at best specific to closely related species (Huang et al., 2009; Liu et al., 2018). In addition, the standardization process involves a series of interconnected steps.
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Internal fertilization makes it difficult to study sperm cryopreservation of live-bearing fishes. Typically, sperm are transferred into the female reproductive tract by artificial insemination [81]. After transfer, sperm bundles must dissociate and spermatozoa must be active to traverse the female reproductive tract.
Germplasm repositories are a necessary tool for comprehensive conservation programs to fully preserve valuable genetic resources of imperiled animals. Cryopreserved germplasm can be used in the future to produce live young for integration into other conservation projects, such as habitat restoration, captive breeding, and translocations; thus compensating for genetic losses or negative changes that would otherwise be permanent. Although hundreds of cryopreservation protocols for various aquatic species have been published, there are great difficulties in moving such research forward into applied conservation projects. Successful freezing of sperm in laboratories for research does not guarantee successful management and incorporation of genetic resources into conservation programs in reality. The goal of the present review is to provide insights and practical strategies to apply germplasm repositories as a real-world tool to assist conservation of imperiled aquatic species. Live-bearing (viviparous) fishes are used as models herein to help explain concepts because they are good examples for aquatic species in general, especially small-bodied fishes. Small live-bearing fishes are among the most at-risk fish groups in the world, and need urgent conservation attention. However, development of germplasm repositories for small live-bearing fishes is challenged by their unusual reproductive characteristics, such as formation of sperm bundles, initiation of spermatozoa motility in an isotonic environment, internal fertilization and gestation, and the bearing of live young. The development of germplasm repositories for goodeids and Xiphophorus species can provide examples for addressing these challenges. Germplasm repositories must contain multiple basic components, including frozen samples, genetic assessment and information systems. Standardization and process generalization are important strategies to help develop reliable and efficient repositories. An ideal conservation or recovery program for imperiled species should include a comprehensive approach, that combines major concerns such as habitat (by restoration projects), population propagation and maintenance (by captive breeding or translocation projects), and preservation of genetic diversity (by repository projects). In this context, strong collaboration among different sectors and people with different expertise is a key to the success of such comprehensive programs.
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Citation Excerpt :
There were no significant differences in post-equilibration sperm motility when DMSO, methanol, and glycerol were used, and for fresh sperm controls, indicating that differences in post-thaw sperm motility when using DMSO and glycerol were attributed to the differential protective effect during freezing and thawing, and not to toxic effects at room temperatures. There were no significant differences in post-thaw sperm motility with use of 5%, 10%, and 15% DMSO, or at 2 min, 6 h, and 24 h after thawing, suggesting that sperm from X. eiseni were not sensitive to the concentration of DMSO within this range, and once the sperm survived freezing and thawing sperm could survive the presence of DMSO for 24 h. Post-thaw sperm motilities were 50% to 70% for poeciliids (Huang et al., 2004c; Yang et al., 2007b; Huang et al., 2009; Yang et al., 2009, 2012a,b), which was greater than the 2% to 9% observed in the present study. This could be because of the relatively less quality of fresh sperm used in the present study with initial motilities of 29% to 34% compared to the 70% to 90% initial motilities used in the cryopreservation of poeciliids.
Previous studies of sperm cryopreservation of livebearing fish have been limited to two genera within the family Poeciliidae. The goal of the present study was to investigate the feasibility to produce live young of livebearing goodeids (family Goodeidae) with cryopreserved sperm, using aquarium-trade populations of the endangered species Redtail Splitfin (Xenotoca eiseni, Rutter, 1896). Reproductive condition of females was evaluated by histological categorization of ovarian development. A total of 117 females were inseminated with cryopreserved sperm, 81 were inseminated with fresh sperm, 27 were mixed with males for natural breeding, and 30 were maintained without males or insemination. Histological images of 34 mature females indicated 68% of ovaries had primary- or secondary-growth oocytes, and 32% had ovulated eggs. Ovarian development had no significant relationship (P =  0.508) with body wet weight, but had a relationship (P <  0.001) with ovary weight and gonadosomatic index. Sperm cells were observed within ovaries that were fixed at 12 h after insemination with fresh sperm. A total of 29 live young were produced from two females inseminated with thawed sperm (8% post-thaw motility with HBSS300 as extender, 20 min incubation in 15% DMSO, cooling rate at 10 °C/min, and thawing at 40 °C for 7 s), 12 were produced from two females with fresh sperm (1%–20% motility), 41 were produced from five naturally spawned females, and no live young were produced from the female-only group. This study provides a foundation for establishment of germplasm repositories for endangered goodeids to assist conservation programs.

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^☆: This work was supported in part by funding from the Qianjiang Talent Plan of Zhejiang Province (No. 2008R10034) and the National Natural Science Foundation of China (No. 30800845).

¹: The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint first authors.

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Sperm cryopreservation in guppies and black mollies—A generalized freezing protocol for livebearers in Poeciliidae☆

Abstract

Introduction

Section snippets

Sperm collection and processing

Effect of cryoprotectant toxicity before freezing

Discussion

Conflict of interest

Acknowledgment

Aquaculture

Aquaculture

Comp. Biochem. Physiol.

Fertil. Steril.

Theriogenology

Cryobiology

Theriogenology

Am. J. Hum. Genet.

Theriogenology

Cryobiology

Methods in Reproductive Aquaculture: Marine and Freshwater Species (Marine Biology)

Cryopreservation of equine sperm: optimal cooling rates in the presence and absence of cryoprotective agents determined using differential scanning calorimetry

Biol. Reprod.

Sperm cryopreservation of live-bearing fishes of the genus Xiphophorus

Combined effect of glycerol concentration and cooling velocity on motility and acrosomal integrity of boar spermatozoa frozen in 0.5 ml straws

Mol. Reprod. Dev.