Poster Presentation IXth International Conference on Boar Semen Preservation 2019

Comparative assessment of respiratory activity in boar and reindeer (Rangifer tarandus) semen (#6.4)

Elena Nikitkina 1 , Artem Musidray 1 , Anna Krutikova 1 , Svetlana Timofeeva 1 , Kirill Plemyashov 1 , Vasilii Goncharov 1 , Ismail Shapiev 1
  1. Russian Research Institute of Farm Animal Genetics and Breeding — Branch of the L.K. Ernst Federal Science Center for Animal Husbandry, St.Petersburg, ST.PETERSBURG, Russia

Disorder related to the metabolism of energy has been linked to a decrease in the fertilising ability of spermatozoa. The amount of ATP present in the cell, as well as the respiration and glycolysis rate are important indicators of energy metabolism. In boars, the process of respiration through oxidative phosphorylation is responsible for the synthesis of ATP, but very little is known about how reindeer sperm generate ATP and energy for sperm processes. The aim of the current work was to compare the cellular respiration rate of boar and reindeer spermatozoa following the addition of 2,4- dinitrophenol (2,4 - DNP) to a semen sample. 2,4 - DNP uncouples proton pumping from ATP synthesis, and should increase respiration rate to indicate efficient oxidative phosphorylation. Semen was collected from 6 boars and 6 reindeer. Following collection, both boar and reindeer semen was divided into 4 treatments (fresh control, fresh+DNP, frozen control, frozen+DNP).  Fresh boar semen was diluted 1:1 either in GHUJKUM medium (inventor's certificate No. 540634, Russia) or GHUJKUM medium suppled with DNP and then assessed.  Boar semen destined for freezing was diluted 1:1 in a similar medium and cooled to 22 °C for 2 h.  It was then centrifuged for 15 min at 700 x g, the supernatant removed, and pellet resuspended to a final concentration of 1.5 billion spermatozoa/ml and cooled to 4-5 °C at 0.2 °C/min. Prior to freezing in the pellets, glycerol was added at a final concentration of 2%. Fresh reindeer semen was extended in either Steredyl or Steredyl supplemented with 2,4 - DNP to a final concentration of 100 million spermatozoa/ml and assessed.  Reindeer semen destined for freezing was extended in a similar medium and allowed to equilibrate in a cold room to 5 °C before being loaded into 0.25 ml straws. Straws were frozen in liquid nitrogen vapour for 10 minutes before being submerged. Cellular respiration rate was assessed by the polarographic method using a Clark electrode. A large difference in the rate of cellular respiration was observed between fresh treatments (P<0,01). Cellular respiration rate after the addition of 2,4-DNP to fresh boar semen increased from 1.6 to 4.1 times (3.2 ± 0,25, mean ± SD).  Similarly, in fresh reindeer semen, cellular respiration rate increased from 1.0 to 2.3 times (1.5 ± 0,49). There was also a difference in respiratory response between frozen-thawed boar and reindeer spermatozoa after the addition of 2,4-DNP (P<0,01). Cellular respiration increased 2.3 ± 0.36 times in frozen-thawed boar sperm but decreased 1.3 ±0.42 times in frozen-thawed reindeer sperm. It is possible that glycolysis prevails in reindeer spermatozoa. The results of this preliminary study suggest further work is needed to understand cellular respiration in reindeer sperm during preservation.

(The authors acknowledge financial support from Russian Science Foundation, Grant No:17-16-01023).