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

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 × 106 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 × 106 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.The German Ministry of Education and Research (BMBF Number 033L046) and by a grant of the German Research Council to J. S. and K. M. (DFG SCHI 476/12–1&2 and MU 1520/4–1&2).http://www.theriojournal.com2018-02-27hj2017Paraclinical Science

Modification of sperm fatty acid composition during epididymal maturation in bats.

Biochemical properties of polyunsaturated fatty acids (PUFAs) are fundamental to sperm movements. Amongst all adjustments operated during epididymal maturation, sperm membrane lipid composition is remodelled. Specifically, the proportion of PUFAs usually increases from the caput towards the cauda epididymidis. In mammals, PUFAs are predominantly acquired through the diet, which can consequently impact male fertility. We aimed at analysing to what extent n-6 and n-3 PUFAs are incorporated into sperm in the Seba's short tailed bat (Carollia perspicillata), and at demonstrating the effect of the sperm fatty acid composition on sperm mobility. We therefore provided food varying in fatty acid composition to males of C. perspicillata, and measured the fatty acid composition and mobility traits in spermatozoa collected from the caput and cauda epididymides. We found that n-6 and n-3 PUFAs and saturated fatty acids were significantly related to sperm velocity but not to the proportion of progressive sperm (i.e. motility). Concomitant to an increase in sperm velocity, the level of fatty acid saturation increased from the caput to the cauda epididymidis, while the proportion of PUFAs remained similar along the epididymis. A reduction in n-6 PUFAs counterbalanced an increase in n-3 PUFAs. The food treatments did not affect the sperm fatty acid composition. Our results suggest that a precise endogenous control rather than dietary effects determines sperm fatty acid composition in C. perspicillata

Low temperature preservation of porcine semen: influence of short antimicrobial lipopeptides on sperm quality and bacterial load

Antimicrobial resistance is a steadily increasing problem and poses a serious threat to global public health. Therefore, it is highly necessary to advance the development of novel antimicrobial compounds and semen preservation strategies. The aim of this study was to evaluate a low temperature, antibiotic-free preservation procedure using Androstar Premium (ASP) extender (Minitüb) with antimicrobial lipopeptides. Firstly, seven lipopeptides in two concentrations (1 × minimum inhibitory concentration (MIC)/2 × MIC) were tested on their sperm-compatibility at 17 °C. Two lipopeptides, C16-KKK-NH2 and C16-KKKK-NH2, did not negatively affect sperm quality and were further evaluated for their efficiency of bacterial growth inhibition at 5 °C. Besides an overall diminution of colony forming units, both peptides showed a reduction of bacterial subcultures (n = 103) with a decrement in Gram-positive rods from 65 (ASP w/o supplements) to 39/52 (ASP w/ C16-KKK-NH2/C16-KKKK-NH2), in Gram-positive cocci from 21 to 9/10 and in Gram-negative species from 17 to 8/5 total subcultures. Furthermore, lipopeptides revealed activity towards selected bacteria of potential concern in artificial insemination like Trueperella pyogenes, Alcaligenes faecalis, Pseudomonas aeruginosa (not C16-KKK-NH2), Pasteurella sp., Providencia stuartii, Escherichia coli (not C16-KKKK-NH2) and Streptococcus porcinus (not C16-KKKK-NH2). Consequently, both tested lipopeptides are promising candidates for alternative antibiotic-free preservation techniques of boar semen