Abstract
Semen cryopreservation in South American camelids has a low efficiency. Post-thaw viability of sperm is low, and poor results are obtained when artificial insemination is performed with cryopreserved semen, impeding advances both in accelerated genetic progress and selection. This study aimed to describe the effect of a conventional method of camelid semen cryopreservation on the llama sperm ultrastructure during cooling and freezing, using transmission and scanning electron microscopy (TEM, SEM). Sperm motility, vigor, viability, and DNA integrity during those steps were also examined. Ejaculates from five fertile adult llama males were obtained by electroejaculation. For cooling, semen samples were washed with Hepes-balanced salt solution (HBSS), diluted in Tris-citric acid-fructose egg yolk extender (TCF-EY), and then cooled until 5°C for 24 h. For freezing, sperm samples were washed with HBSS, diluted in TCF-EY and cooled until 5°C for 2.5 h. Samples were equilibrated with TCF-EY, supplemented with 6% glycerol at 5°C for 20 min, and then stored in liquid nitrogen for a month before thawing. TEM and SEM analyses were carried out on sperm samples prior to cryopreservation, after cooling down until 5°C for 2.5 and 24 h, and after the freeze-thaw process. Ultrastructural injury was noticed during cooling, even though sperm motility, vigor, viability, and DNA integrity were not significantly affected. Analysis revealed plasma membrane and acrosome damage, loss of mitochondria, and axoneme and periaxonemal structure disorganization after 2.5 h of cooling. During freezing, a significant decrease in sperm motility and viability was observed after thawing. TEM and SEM revealed prominent signs of post-thawing damage. The plasma membrane was lost or exhibited various degrees of swelling, undulation, and perforations. Besides, the sperm presented vacuoles in the nucleus and broken acrosomes. Mitochondria in the midpiece showed vacuolization and structural disorganization. In conclusion, SEM and TEM revealed that cryopreservation induced ultrastructural damages in llama sperm that initiated during cooling and intensified during freezing. These details provide valuable data for further studies to minimize cryodamage in camelid sperm.
Highlights
Semen cryopreservation is a widely used technique to preserve and supply sperm for breeding and maintenance of genetic diversity in wildlife
Concerning sperm motility, vigor, viability, and DNA integrity, no differences were found between C0 and C24 sperm samples during cooling (Tables 2, 3)
The freezing procedure resulted in a significant reduction in spermatozoa viability after thawing (FT) compared with F0 and F2.5 values (p < 0.05), but no differences were detected regarding DNA integrity (Table 4)
Summary
Semen cryopreservation is a widely used technique to preserve and supply sperm for breeding and maintenance of genetic diversity in wildlife. During a standard cryopreservation protocol, different processing steps are involved (dilution of semen at 37◦C with the extender and cooling until 5◦C, addition of a cryoprotectant and equilibration, and freezing in liquid nitrogen at −196◦C), and it is not entirely clear how each step affects the sperm cell [1]. It has been shown that cryopreservation induces deleterious alterations in sperm structure and function [2,3,4,5]. These involve thermal stress due to the change in temperature during cooling, freezing and thawing, as well as osmotic stress caused by the addition of high concentrations of cryoprotective agents and crystallization [6]. Llama and alpaca pregnancy rates with frozen-thawed semen ranged from 0 to 26% [9,10,11,12], while maximum pregnancy (33%) was obtained with cooled semen at 5◦C [14]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
