Gamete quality and management for in vitro fertilisation in meagre (Argyrosomus regius)

Abstract

The aquaculture of meagre (Argyrosomus regius) requires methods for the control of reproduction that enable the production of families from specific individuals for selective breeding programs. We experimentally determined the parameters required for an in vitro fertilisation protocol. A total of 14 females and 5 males (mean ± S.D. weights of 20.45 ± 6.22 and 15.94 ± 2.75 kg, respectively) were used. Selected females had vitellogenic oocytes >550 μm in diameter and males had fluid sperm upon application of abdominal pressure. Both sexes were treated with an injection of 15 μg kg−1 of gonadotropin-releasing hormone agonist (GnRHa) to induce oocyte maturation/ovulation and enhance sperm production. To determine the timing of ovulation and window of high egg viability, females were stripped serially every 2.5 h beginning 35 h after GnRHa treatment. Sperm was obtained 24 h after GnRHa treatment and was diluted 1/4 in modified Leibovitz for storage at 4 °C until use. Sperm quality parameters such as percentage initial spermatozoa motility, duration of motility, velocity and density were determined using computer assisted sperm analysis (CASA). In vitro inseminations were made in duplicate or triplicate batches of eggs from each spawn by mixing 0.5-1 mL of eggs, 20-40 μL diluted sperm (pooled from two males) and 100 mL of seawater. Fertilisation success was examined at spermatozoa (spz): egg ratios between ~2000 and 400,000 spz egg−1. The optimal time for stripping ovulated females was ≤3 h after ovulation, which was the window of optimal egg viability. Ovulation under the conditions of this study was close to 38 h after GnRHa treatment, with a range from 35 to 41 h. Beginning from 3 h after ovulation, egg viability declined probably due to overripening. Sperm diluted in Leibovitz maintained motility and velocity for as long as 7 h after collection. Spermatozoa motility (%) and average path velocity (VAP, μm/s) of sperm samples obtained from males before GnRHa injection declined rapidly after activation compared to the samples obtained 24 h post-injection, with significant decreases respectively after 75 and 45 s. A minimum ratio of 150,000 spermatozoa egg−1 was necessary to ensure high fertilisation success. The acquired knowledge of the present study will aid the aquaculture industry and future research on selective breeding programs for meagre. Highlights ► An in vitro fertilisation protocol has been developed for meagre (Argyrosomus regius). ► Ovulation took place close to 38 h and in the range of 35-41 h after GnRHa induction at 18 °C. ► Good quality eggs were collected within a three-hour window from ovulation. ► The spermatozoa: egg ratio for high fertilisation success was 150,000 spermatozoa egg −1 .

Low sperm to egg ratio required for successful in vitro fertilisation in a pair-spawning teleost, Senegalese sole (Solea senegalensis)

“…Senegalese sole sperm has variable and generally poor quality with low percentage motility (González-López et al, 2020). In the present study as in other studies (Ramos-Júdez et al, 2019), the effect that percentage motility has on the spz egg -1 ratio was removed by examining the relationship between the number of motile sperm and eggs fertilised. Many studies simply express total number of sperm (including immotile sperm) in the spz egg -1 ratio, however, this is inaccurate and should be stated with the percentage motility of the sperm used.…”

“…Egg quality appeared to decline during the first 30 min of storage with no plateau period of good egg quality, which indicated the sooner eggs were fertilised after stripping the higher the viability of the eggs. This decline in egg quality after ovulation and stripping has been described in a wide range of species as the overripening process, where eggs age and in association with morphological and biochemical changes lose viability and fertilisation rates decline (Mañanós et al, 2008;Ramos-Júdez et al, 2019;Samarin et al, 2011). A rapid decline in egg quality has been observed in other species, but the decline was initiated after a period of good egg quality of 1 h in curimata (Prochilodus marggravii) (Rizzo et al, 2003) and 50 min in meagre (Argyrosomus regius) (Ramos-Júdez et al, 2019).…”

Low sperm to egg ratio required for successful in vitro fertilization in a pair-spawning teleost, Senegalese sole ( Solea senegalensis )

“…Egg quality appeared to decline during the first 30 min of storage with no plateau period of good egg quality, which indicated the sooner eggs were fertilized after stripping the higher the viability of the eggs. This decline in egg quality after ovulation and stripping has been described in a wide range of species as the overripening process, where eggs age and in association with morphological and biochemical changes lose viability and fertilization rates decline [ 17 , 43 , 44 ]. A rapid decline in egg quality has been observed in other species, but the decline was initiated after a period of good egg quality of 1 h in curimata ( Prochilodus marggravii ) [ 45 ] and 50 min in meagre ( Argyrosomus regius ) [ 43 ].…”

“…This decline in egg quality after ovulation and stripping has been described in a wide range of species as the overripening process, where eggs age and in association with morphological and biochemical changes lose viability and fertilization rates decline [ 17 , 43 , 44 ]. A rapid decline in egg quality has been observed in other species, but the decline was initiated after a period of good egg quality of 1 h in curimata ( Prochilodus marggravii ) [ 45 ] and 50 min in meagre ( Argyrosomus regius ) [ 43 ]. There is considerable variation across species and some species have very different egg storage capacities, for example, eggs from a Cyprinidae species kutum ( Rutilus frisii ) [ 44 ] maintained good egg quality during 8 h of storage and salmonid eggs can be stored successfully for 4–5 days [ 40 ].…”

“…Senegalese sole sperm has variable and generally poor quality with low percentage motility [ 22 ]. In the present study as in other studies [ 43 ], the effect that percentage motility has on the spz egg −1 ratio was removed by examining the relationship between the number of motile sperm and eggs fertilized. Many studies simply express total number of sperm (including immotile sperm) in the spz egg −1 ratio, however, this is inaccurate and should be stated with the percentage motility of the sperm used.…”