Biology of sperm and artificial reproduction in carp

doi:10.1016/0044-8486(94)00231-C

Aquaculture

Volume 129, Issues 1–4, January 1995, Pages 95-112

https://doi.org/10.1016/0044-8486(94)00231-C Get rights and content

Abstract

Although the common carp, Cyprinus carpio, has been cultivated for several thousand years and is produced in large quantities, research on reproduction has been very limited. Traditionally, spawning occurred naturally in situ in rearing ponds. In slightly improved methods large breeding ponds stocked with brood fish were devoted to reproduction with fry collection in autumn, or in small spawning ponds with collection of larvae a few days after hatching. Controlled reproduction in hatcheries started only in the 1950s. This paper reviews some of the basic work on carp sperm and describes the technologies of artificial reproduction. The sperm is of a primitive type with uncondensed chromatin and a small midpiece. As in most teleost fish, the sperm is immotile in the male genital tract and in the semen and is activated after release into fresh water. The initiation of motility is due to the decrease in osmotic pressure. The structure of the flagellum is rapidly disorganized in fresh water and sperm stop moving after 30 s. When dilution occurs in a 50 mM NaCl solution, the osmotic change is sufficient to initiate motility, but the flagellum is not disorganized and swimming lasts a few minutes. Motility depends mainly on endogenous ATP stores, about $12 nmol 10^{8}$ spermatozoa, and stops when 50–80% of the ATP is exhausted by hydrolysis. The procedure of artificial insemination includes collection of gametes (from “hypophysised” males and females), mixing in an extender (45 mM NaCl, 5 mM KCl, Tris 2.5 mM, glycine 19 mM, pH 8) in a ratio 1 litre of eggs to 1 litre of diluent and 1 ml of semen. Sperm motility can be triggered during collection by contamination of the semen with urine; this could interfere with fertilization and, if present, urine should be discarded by pouring out the top part of the tube. The sticky layer of the egg is removed by adding a “dissolving solution” (20 g urea/1 + 4g NaCl/1 of water) or milk (diluted $15$ in water) to the fertilized egg and stirring. One hour later the swollen eggs are transferred to incubators (usually Zug bottles, sometimes 200 litre circular jars).

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This study emphasizes the need for sperm quality assessment and cryopreservation strategy in the spotted scat Scatophagus argus as the occurrence of male population of this species is limited. Analyses of sperm quality were made including ionic content and osmolality of seminal plasma and blood serum. Sodium chloride (0.9%) was optimized as the extender with a dilution ratio of 1:3 (milt:cryomedium) for all experiments. Cryoprotectants including dimethyl sulfoxide (DMSO), dimethyl acetamide (DMA), glycerol (GLY), propylene glycol (PG), ethylene glycol (EG) and methanol (MeOH) were screened at various concentrations and equilibration times. Cryopreservation was performed using the cryoprotectants DMSO, GLY, EG, and MeOH individually as well as in combinations. Experiment 1 was done with DMSO, GLY, EG and MeOH (5, 10 and 15%); experiment 2 was done with DMSO (5%) + GLY (5%), DMSO (5%) + EG (5%), DMSO (5%) + MeOH (5%). Simultaneously, experiment 3 was done with both cryoprotectants and co-cryoprotectants viz. DMSO (5%) + egg yolk (5%), DMSO (5%) + coconut water (5%), DMSO (5%) + tomato water (5%) and DMSO (5%) + honey (5%). The cooling sample was optimized and performed with a one-step freezing protocol of −5 °C/min from 25 °C to −150 °C. The results indicated that samples subjected to experiment 2 using DMSO (5%) + EG (5%) for 60 days of storage registered maximum motility (82.33 ± 2.51%), duration time of sperm motility (10.45± 1.01 min) and viability (84.14 ± 5.29%) of post-thaw cryopreserved sperm. Morphology and ultrastructure of post-thaw sperm showed smooth surface topography and normal architecture of cellular organelles. Biochemical analysis indicated a reduction in the protein content of post-thaw cryopreserved sperm. DNA integrity (Comet assay) showed 6.30 ± 1.00% of tail DNA in post-thaw sperm, whereas fresh sperm registered 2.41 ± 1.85%. Functional integrity using the HOST of post-thaw sperm showed a maximum of 82.66 ± 1.15% with DMSO (5%) + EG (5%). Finally, fertility of post-thaw sperm using mature eggs of wild-caught females indicated the entry of sperm by breaking the vitelline membrane. Overall it was demonstrated that the DMSO (5%) + EG (5%) was amenable to the long-term preservation of sperm. Therefore, this protocol can be used as an alternative for male broodstock development under captive breeding program.
Aging of common carp (Cyprinus carpio L.) sperm induced by short-term storage does not alter global DNA methylation and specific histone modifications in offspring
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Short-term storage of sperm is a useful tool to synchronize the availability of male and female gametes for fertilization in a hatchery. Induced aging by short-term storage has been shown to affect fish spermatozoa phenotypes including morphology and motility kinetics. However, its effects on epigenetics of sperm and the resulting embryos have not been investigated. In the present study, sperm of common carp (Cyprinus carpio) was stored in vivo and/or in vitro using an extender, and spermatozoa motility kinetics, viability, and epigenetics were analyzed. We observed that spermatozoa motility and velocity decreased following in vivo and/or in vitro storage from 3 to 6.5 days, but spermatozoa viability remained stable. At the same time, global DNA methylation, DNA hydroxymethylation, and specific histone acetylation and methylation were not affected by sperm aging. Further analysis showed no variation in global DNA methylation during embryogenesis when short-term stored sperm in the extender was used for fertilization compared with embryos produced from fresh sperm. The results suggest the reliability of short-term storage of sperm using an extender in fertilization and the production of the next generation of embryos. However, larval growth and performance need to be elucidated for these fish. This study has shed light on the potential application of extenders in storage of sperm in common carp breeding programs to manage selection, genomic manipulation and genetic resources conservation.
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This is the first report showing fertilization of short-term stored eggs (19 °C) with short-term stored diluted sperm (0–2 °C) in common carp (Cyprinus carpio). From 5 males, fresh (control) and 7-day stored sperm were diluted with common carp extender. After collection of eggs from nine females, the eggs were immediately pooled into three groups from females 1–3, 4–6 and 7–9, according to the eggs' quality based on visual criteria. They were placed in beakers, covered and incubated at 19 °C (for each pool, 10 g of eggs from each female were used). The 7-day short-term storage sperm was incubated at 20 °C for 20 min and pooled before being used for fertilization. The eggs were fertilized with fresh and short-term storage sperm after 1, 3 and 6 h post storage of eggs and activated with hatchery water and Perchec solution. Sperm motility and velocity (50–54% and 80–115 µm/s) of 7-day short-term storage sperm at 0–2 °C were ensured by additional incubation of sperm at 20 °C for 20 min before being activated. Principal component analysis showed that the sorting of eggs of different females into three groups according to sperm quality, fertilization ability and production of malformed larvae was successful. It is recommended that overripe common carp eggs are stored for a maximum of 1 h. A period of 3 h can be used for better quality eggs and exceptionally, up to 6 h for very high-quality eggs. Higher larvae malformation was found in the 6 h aged eggs together with fertilized short-term storage and fresh sperm. In all quality groups of eggs, the effect of different sperm, activation medium and egg storage time on the level of malformations was observed. The activation solution for carp did not show any positive fertilization and hatching effects.
Elevated temperature promotes spermatozoa motility kinetics and fertilizing ability following short-term storage: An implication for artificial reproduction of common carp Cyprinus carpio in a hatchery
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Citation Excerpt :
Sperm short-term storage is a simple and practical biological tool, which has been widely applied in hatchery management in aquaculture (Stoss, 1983; Cabrita et al., 2008; Contreras et al., 2020). It involves dilution of fresh sperm in an extender to maintain spermatozoa in a quiescent state with minimal metabolic activity, and storage at a cool temperature for hours to weeks while spermatozoa maintain their functional properties of motility and fertilizing ability (Billard et al., 1995; Zietara et al., 2009; Contreras et al., 2020). Recently, a high fertilization rate was reported for common carp (Cyprinus carpio L.) sperm stored without antibiotics in an extender for 6 days at 0–2 °C (Cheng et al., 2022) and in an extender with antibiotics for 14 days at 10 °C (Cejko et al., 2022).
Short-term storage of sperm in an extender at a low temperature has been widely applied in hatchery management. In the present study, for the first time, we examined the effects of elevating temperature on spermatozoa motility kinetics and fertilization ability after short-term storage at 0–2 °C. Sperm of common carp (Cyprinus carpio L.) was diluted with an extender (110 mM NaCl, 40 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 20 mM Tris, pH 7.5, 310 mOsmol/kg) at the ratio 1:1 (v/v) in containers and stored for 6–9 days at 0–2 °C under aerobic conditions. At the end of storage, the sperm suspension was incubated at 20 °C for >10 min and spermatozoa motility, velocity [straight-line velocity (VSL) and curvilinear velocity (VCL)], viability, and fertilizing ability were recorded. Spermatozoa motility of sperm stored for 6 days was significantly increased after incubation at 20 °C for 10 and 20 min and activation in both distilled water and a saline solution (45 mM NaCl, 5 mM KCl, 30 mM Tris, pH 8.0). Elevating the temperature did not affect spermatozoa viability. Spermatozoa motility, VSL and VCL, and viability were decreased at 3 days post storage (DPS) after activation in distilled water without incubation at 20 °C. However, elevating the temperature for 10–20 min at any time post sperm storage (3, 6 or 9 DPS) could promote spermatozoa motility kinetics. For sperm stored for 9 days, fertilization and hatching rates were increased when it was incubated at 20 °C for 10 and 20 min prior to fertilization at the ratio 30,000 spermatozoa per egg. Increasing the spermatozoa to egg ratio to 100,000:1, however, masked the temperature effects on fertilization and hatching rates. Finally, a large-scale experiment was performed in hatchery in which sperm stored for 8 days was incubated for 2 h at 20 °C, resulting also in an increase in spermatozoa motility and fertilizing ability. Results of the present study show that elevating the temperature following short-term storage of common carp sperm at low temperatures improved spermatozoa motility kinetics and their fertilizing ability. This method may contribute to developing artificial reproduction of economically important fish species in aquaculture.

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Biology of sperm and artificial reproduction in carp

Abstract

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Biol. Cell.

Aquaculture

Aquaculture

Aquaculture

Aquaculture

Comp. Biochem. Physiol.

Aquaculture

Aquaculture

Aquaculture

Aquaculture

Aquaculture

Aquaculture

Aquaculture

Biochim Biophys. Acta

Aquaculture

Aquaculture

Détermination du rôle des protéines du liquide séminal dans l'aptitude à la motilité des spermatozoïdes de carpe

Biosynthesis of 17α,20α-dihydroxy-4-pregnen-3-one from 17 α-hydroxyprogesterone by spermatozoa of the common carp, Cyprinus carpio

J. Exp. Zool.

Fish germinal cells. I. Comparative spermatology of seven cyprinid species

Gamete Res.

Effects of 17α,20β-dihydroxy-4-pregnen-3-one on the in vitro production of 11-ketotesterone by testicular fragments of the common carp, Cyprinus carpio

J. Exp. Zool.

The shift from C-19 to C-21 steroid synthesis in spawning male common carp, Cyprinus carpio, is regulated by the inhibition of androgen production by progestogens produced by spermatozoa

Biol. Reprod.

The mechanism regulating the shift from androgen to progestogen production during spawning in male common carp (Cyprinus carpio): a review

Ecological-physiological peculiarities of semen of pond carps. II. Change in the physiological parameters of spermatozoids of some carps under the influence of environmental factors

J. Ichthyol.

Ecological and physiological properties of semen of pond cyprinids. III. Physiological-biochemical parameters of the semen of some cyprinid species

J. Ichthyol.

Ultrastructure comparée de spermatozoïdes de quelques poissons téléostéens

Quaderno

Utilisation d'un système Tris-glycocolle pour tamponner le dilueur d'insémination pour truite

Bull. Fr. Pisc.

Spermatogenesis and spermatology of some teleost fish species

Reprod. Nutr. Dév.

Artificial insemination and gamete management in fish

Mar. Behav. Physiol.

Artificial insemination in fish

The energetics of fish sperm motility

Some problems related to the assessment of sperm motility in freshwater fish

J. Exp. Zool.

Biology of gametes, eggs and embryos

Testicular and some hormonal changes during the first four years of life in the mirror carp, Cyprinus carpio L.

J. Fish Biol.

Mise en évidence de quelques enzymes dans le sperme de la carpe Cyprinus carpio L. et de la truite Salmo gairdneri Richardson et dans le liquide coelomique de la truite

CR Acad. Sci. Paris

Producción intensiva de carpia en la isla de Mallorca (sistema integrado en usos agricolas convencionales, con recirculación de agua, al aire libre)