The current status of sperm cryopreservation of the endangered Probarbus jullieni (Sauvage) in Malaysia

Abstract

Summary The objective of this study was to develop a cryopreservation method in Probarbus jullieni sperm, an endangered riverine fish species in Southeast Asia, including the optimization of an extender solution (14 extender formulations were tested) and selecting a cryoprotectant (five types of agents and methanol were used at concentrations (v/v) of 5, 7.5, 9, 10, 12, 15 and 20%). The semen to diluent ratios tested were as follow: 1 : 1, 1 : 2, 1 : 3, 1 : 4, 1 : 5, 1 : 7, 1 : 9, 1 : 14, 1 : 19, 1 : 24 and 1 : 49. Vapour exposure duration was set at 5, 10, 15 and 20 min while the distance between sample and liquid nitrogen (LN2) during the vapour exposure was designed at 3, 3.5, 4, 5 and 6 cm. Further, the time frame for thawing was set at 6, 7, 8, 10, 20 and 30 s. The optimum protocol was by using CF‐HBSS (pH 7.5, osmolality 285 ± 10 mOsmol kg−1) in combination with methanol at 9% (v/v); sperm to diluents ratio between 1 : 3 to 1 : 5; vapour exposure for 5 min or 10 min, with samples placed at 3.5 cm or 4 cm above LN2 and thawing at 40°C for 7 s. The mean of pre‐frozen and post‐thaw sperm motility was 80.1 ± 13.6% (n = 43) and 49.6 ± 16.4% (n = 43) respectively. The reproductive characteristics of P. jullieni during its spawning season were addressed in present work. Cryopreserved sperm was found to have lower fertilization ability (4.2 ± 2.5%, n = 1050) and hatching rate (1.6 ± 1.2%, n = 1050) compared with fresh sperm (fertilization 77.7 ± 6.2%, n = 1050; hatching 64.7 ± 7.7%, n = 1050). The resulted problems and constraints encountered in the process of sperm cryopreservation of the species studied were also reported in this paper.

Preservation of black sharkminnow,Labeo chrysophekadion(Bleeker, 1849) spermatozoa

“…Osmolality is also a critical variable in sperm quality affecting performance. The osmolality (mOsmol kg À1 ) of L. chrysophekadion seminal plasma (244.0-258.0) coincided with the seminal plasma osmolality of Cyprinidae, which was within 230.0-346.0 such as 254.0-267.0 in Alburnus alburnus (Lahnsteiner, Berger, Weismann & Patzner 1996), 236.0-318.0 in V. vimba (Alavi, Kozak et al 2010) and 246.0-313.0 in Probarbus jullieni, (Chew et al 2010) (Table 6), but these osmolalities were slightly lower than the other cyprinid species such as B. barbus (268.0-276.0) and B. sharpeyi (274.5 AE 9.0) reported by Alavi, Psenicka et al (2008) and Alavi, Jorfi et al (2010), respectively, 264.3-267.0 in C. carpio (Cejko et al 2014). pH seminal plasma of black sharkminnow (8.10) was slightly alkaline and is similar to the other cyprinid fish such as, C. carpio (7.53-7.67;Kruger, Smith, Van Vuren & Ferreira 1984; Semen was diluted with each extender and stored at 4°C for five days.…”

“…) and 246.0–313.0 in Probarbus jullieni , (Chew et al . ) (Table ), but these osmolalities were slightly lower than the other cyprinid species such as B. barbus (268.0–276.0) and B . sharpeyi (274.5 ± 9.0) reported by Alavi, Psenicka et al .…”

“…) , Puntius gonionotus (7.00–7.50; Routray, Dash, Dash, Swain, Sarkar & Sarangi ), P. jullieni (7.14–7.85; Chew et al . ), Rutilus rutilus caspicus (7.45–7.95; Golpour et al . ) and L. calbasu (7.56; Hassan et al .…”

“…This study was similar to that of Chew et al . (), who reported that isotonic osmolality of the extender used (calcium‐free Hank's balance salt solution, CF‐HBSS) yielded a higher motility (69.3 ± 5.9%) than other extenders ( P < 0.05) and was chosen for cryopreservation of P. jullieni sperm. Similarly, in the case of silver barb, P. gonionotus , the mimicking media of extender‐4 was used for cryopreservation of silver barb spermatozoa due to its similar seminal plasma to spermatozoa (Routray et al .…”

“…Temperature and pH are also major factors affecting sperm motility (Alavi & Cosson 2005a). Preservation of spermatozoa using different extenders has been successfully reported in some cyprinid fish, such as Kurokura solution (KU) (Rodina, Cosson, Gela & Linhart 2004), modified Cortland solution (MC) (Singsee, Imsilp, Pewnane & Sukumasavin 2005), extender I, 300 mM glucose (Bozkurt, Ogretmen & Secer 2009), Hanks' balanced salt solution (HBSS) (Jing, Huang, Bai, Tanguay & Dong 2009), calcium-free Hanks' balanced salt solution (CF-HBSS) (Chew, Hassan, Rashid & Chuah 2010) and Alsever's solution (Hassan, Nahiduzzaman, Mamun, Taher & Hossain 2014). The extender solution should be compatible with the seminal plasma of the candidate species.…”