Semen cryopreservation and radical reduction capacity of seminal fluid in captive African lion (Panthera leo)

Abstract

Optimizing cryopreservation protocols for nondomestic felids contributes to the successful development of assisted reproduction techniques and genetic resource banking. In this study, we describe a simple cryopreservation procedure for African lion (Panthera leo) ejaculates, which was tested with different packaging options and different sperm numbers per dose. By applying urethral catheterization and electroejaculation, 17 ejaculates with greater than 20% motile and greater than 5% progressively motile sperm were collected. A lyophilized extender (a modified egg yolk-Tes-Tris-fructose-glycerol medium) was rehydrated and added in one step at ambient temperature (∼25 °C) to semen, which was prediluted in cell culture medium M199. After slow cooling of insulated samples to 15 °C in a refrigerator (4 °C), the samples were fast frozen over the surface of liquid nitrogen or in a dry shipper. Aliquots of 300 μL containing 20 × 10 sperm were frozen in cryovials and in 0.5-mL straws. Differences were observed in the total motility after thawing between vial (31.5 ± 14.1%) and straw freezing (20.1 ± 8.6%). However, the subpopulations of vital (22.7 ± 7.8% for vial and 19.8 ± 8.5% for straw) and progressively motile (10.0 ± 7.9% for vial and 10.0 ± 6.4% for straw) sperm after washing and 1 hour incubation at 38 °C were of similar magnitude, velocity, and linearity for both packaging options. After freezing of five ejaculates with 20, 60, and 100 × 10 sperm per dose, best results were achieved at the lowest concentration. In general, post-thaw results were highly variable (2.2% and 56.5% total motility) and not correlated to motility or morphology of the fresh semen. To further characterize semen quality, we assessed the protective potential of seminal fluid against oxidative stress, which might be challenged on freeze thawing. The capacity of seminal fluid to reduce radicals was measured in 10 semen samples by electron spin resonance spectroscopy and a spin-labeled fatty acid as a radical probe. Moreover, we determined the lysophosphatidylcholines (LPC) as potential lipid oxidation products in the sperm and erythrocytes of the males. Individuals with a high radical reduction capacity in the seminal fluid and a low LPC content in their erythrocytes showed a better cryosurvival of sperm. This is a first indication that seminal fluid may affect the freezing potential of African lion ejaculates.

Quality of domestic cat semen collected by urethral catheterization after the use of different alpha 2-adrenergic agonists

“…Semen collection has been performed using urethral catheterization after the administration of an α 2 -adrenergic agonist in cats, but medetomidine drugs are emphasized in several studies; their effectiveness for this procedure is referenced in both domestic and wild cats. 8–24 MED is a selective α 2 -adrenergic agonist composed of the enantiomers DEX and levomedetomidine. 24 DEX is the newest and most selective α 2 -adrenergic agonist used with effects such as sedation, analgesia and anxiolysis.…”

The effects of type I collagenase on the degelification of chimpanzee (Pan troglodytes) semen plug and sperm quality

“…Electroejaculation (EEJ) is widely used for semen collection in wildlife and captive zoo animals [ 10 , 11 ]. However, EEJ in great apes has been discouraged, as it incurs higher risk due to the need for deep anesthetization and the high electric voltage required to induce ejaculation.…”

Assisted reproduction for felid species conservation—Sperm competences at risk

“…Cryopreservation means chemical, osmotic, mechanic and oxidative stress to sperm (Hammerstedt, Graham, & Nolan, 1990;Luther et al, 2017). Notably, neither epididymal nor electroejaculated domestic cat sperm is susceptible to cold shock if cooled at moderate cooling rates to 4°C (Hermansson, 2006) and reveals reasonable freezing results (Klaus et al, 2016).…”

“…In lions, glycerol is tolerated as a cryoprotectant at 5% (vol/vol) and is slightly superior to DMSO even if added to semen at temperatures above 20°C before cooling (Luther et al, 2017). However, glycerol and all the extender components suffice for only approximately 20-60 × 10 6 lion sperm in a semen aliquot of 300 µl (Luther et al, 2017). A further increase in sperm concentration leads to a reduction of sperm quality in frozen-thawed samples.…”

“…We analysed the capacity to reduce free radicals in lion seminal fluid separated from semen collected via urethral catheterization. The variability in cryoperformance was significant between ejaculates from different individuals, and individuals with a high radical reduction capacity in the seminal fluid showed a better cryosurvival of sperm (Luther et al, ). Those individual differences in seminal fluid properties might explain that the effect of extender supplementation by antioxidants is dependent on the individual ejaculate conditions.…”