Fresh, equilibrated and post-thaw sperm quality of Brycon orbignyanus (Valenciennes, 1850) and Prochilodus lineatus (Valenciennes, 1837) treated with either salmon GnRHa and domperidone or pituitary extract

The effects of reduced doses of Ovaprim™ (GnRHa + domperidone) on sperm release of Brycon orbignyanus and Prochilodus lineatus were evaluated. Furthermore, sperm quality was compared among fresh, equilibrated and post-thaw samples. Males received a single and reduced dose of Ovaprim™ (0.125 or 0.25 ml/kg); control males received pituitary extract (cPE; 3 mg/kg). Fresh sperm was evaluated for volume, concentration, seminal plasma osmolality and seminal plasma pH. Then sperm was diluted in a freezing medium, equilibrated for 15-20 min and frozen in nitrogen vapor vessel (dry-shipper). Sperm motility was analyzed during 60 s post-activation in fresh, equilibrated and post-thaw samples. Sperm quality of males treated with Ovaprim™ (both doses) were not different from that of cPE-treated males, thus these data were pooled. In B. orbignyanus, motility was higher in fresh (99%) than in equilibrated sperm (81%); post-thaw motility dropped to 42%. In P. lineatus, motility was similar in fresh (99%) and equilibrated sperm (92%); post-thaw motility was 73%. Motility decreased as a function of time post-activation, and this decrease was significant after 60 s in fresh and equilibrated sperm, and as soon as 30 s in post-thaw sperm, in both species. Ovaprim™ at 1/4 of the recommended dose can successfully replace cPE.

O efeito de doses reduzidas de Ovaprim® (GnRHa + domperidona) na liberação do sêmen de Brycon orbignyanus e Prochilodus lineatus foi avaliado. Além disso, a qualidade do sêmen foi comparada entre as amostras frescas, equilibradas e descongeladas. Os machos receberam dose única e reduzida de Ovaprim® (0,125 ou 0,25 ml/kg); os machos-controle receberam extrato de hipófise (cPE; 3 mg/kg). O sêmen fresco foi avaliado quanto ao volume, concentração, e osmolalidade e pH do plasma seminal. Em seguida, o sêmen foi diluído num meio de congelamento, equilibrado por 15-20 min e congelado em botijão de vapor de nitrogênio (dry-shipper). A motilidade espermática foi analisada durante 60 s pós-ativação no sêmen fresco, equilibrado e descongelado. A qualidade do sêmen não diferiu entre os machos tratados com Ovaprim® (ambas as doses) ou cPE, assim foi feito um pool desses dados. Em B. orbignyanus, a motilidade foi maior no sêmen fresco (99%) do que no equilibrado (81%); a motilidade do sêmen descongelado caiu para 42%. Em P. lineatus, a motilidade foi semelhante entre o sêmen fresco (99%) e equilibrado (92%); a motilidade do sêmen descongelado foi 73%. A motilidade caiu em função do tempo pós-ativação, e essa queda foi significante após 60 s no sêmen fresco e equilibrado, e tão precoce quanto 30 s no sêmen descongelado, em ambas as espécies. Ovaprim® a 1/4 da dose recomendada pode substituir o cPE com sucesso.

Methyl glycol, methanol and DMSO effects on post-thaw motility, velocities, membrane integrity and mitochondrial function of Brycon orbignyanus and Prochilodus lineatus (Characiformes) sperm.

The aim of this study was to use more accurate techniques to investigate the effects of cryoprotectants (CPAs) and extenders on post-thaw sperm quality of Brycon orbignyanus and Prochilodus lineatus. Six freezing media comprising the combination of three CPAs (DMSO, methanol and methyl glycol) and two extenders (BTS and glucose) were used. Sperm was diluted in each medium, loaded into 0.5-mL straws, frozen in a nitrogen vapor vessel (dry-shipper), and stored in liquid nitrogen at -196 °C. Post-thaw sperm motility rate and velocities (curvilinear = VCL; straight line = VSL; average path = VAP) were evaluated using a computer-assisted sperm analyzer. Membrane integrity and mitochondrial function were determined using fluorochromes. Post-thaw quality was considered high when samples presented the following minimum values: 60 % motile sperm, 140 µm/s of VCL, 50 % intact sperm membrane and 50 % mitochondrial function integrity. High post-thaw quality was observed in B. orbignyanus sperm frozen in BTS-methyl glycol and in P. lineatus sperm frozen in BTS-methyl glycol, glucose-methyl glycol and glucose-methanol. All samples frozen in DMSO yielded low quality. The presence of ions in the BTS extender affected post-thaw sperm quality positively in B. orbignyanus and negatively in P. lineatus. Methyl glycol was the most suitable CPA for both fish species, leading to a good protection of cell membrane, mitochondrial function and motility apparatus during the cryopreservation process. For an improved protection, B. orbignyanus sperm should be frozen in an ionic freezing medium.

Gamete quality of streaked prochilod Prochilodus lineatus (Characiformes) after GnRHa and dopamine antagonist treatment.

The efficiency of Ovaprim™ salmon gonadotropin-releasing hormone agonist (GnRHa) and dopamine antagonist on the induction of spawning and spermiation in Prochilodus lineatus in comparison with the commonly used method using pituitary extract (PE) was evaluated. Females received PE at 0.5 + 5.0 mg/kg and Ovaprim™ at 0.05 + 0.45 ml/kg or at 0.125 + 0.375 ml/kg. All males received a first dose of PE at 0.4 mg/kg and then PE at 4.0 mg/kg or Ovaprim™ at 0.25 ml/kg. Oocyte, egg, larvae and sperm quality were evaluated. All females spawned and oocyte, egg and larvae quality were similar between Ovaprim™-treated (both doses) and PE-treated females. Data from females were pooled and the mean values were: 242 g ova weight, 15% ova index, 1209 oocytes/g ova, 284,539 oocytes/female, 183 oocytes/g body weight, 1.18 mm oocyte diameter, 49% fertilization rate, 43% hatching rate and 89% normal larvae. Sperm quality was similar between Ovaprim™-treated and PE-treated males. Data from males were pooled and the mean values of semen were: volume of 3.0 ml, 14.9 × 109 sperm/ml, osmolality of 283 mOsm/kg, pH of 7.4, 71% motile sperm, 217 µm/s curvilinear velocity, 102 µm/s straight-line velocity and 189 µm/s average path velocity. Ovaprim™ treatment can be used for commercial reproduction of P. lineatus, without any loss of gamete quality in comparison with PE treatment.