Evaluation of bull spermatozoa during and after cryopreservation: Structural and ultrastructural insights

Abstract

Semen cryopreservation is a well-established procedure used in veterinary assisted reproduction technology applications. We investigated damaging effects of cryopreservation on the structural and ultrastructural characteristics of bull sperm induced at different temperatures and steps during standard cryopreservation procedure using transmission (TEM) and scanning electron microscopy. We also examined the effect of cryopreservation on sperm DNA and chromatin integrity. Five healthy, fertile Friesian bulls were used, and the ejaculates were obtained using an artificial vagina method. The semen samples were pooled and diluted in a tris-yolk fructose (TYF) for a final concentration of 80 × 106 spermatozoa/ml. The semen samples were packed in straws (0.25 ml), and stored in liquid nitrogen (−196°C). Samples were evaluated before dilution, just after dilution (at 37°C), at 2 h and 4 h during equilibration, and after thawing (37°C for 30 s in water bath). In association with step-wise decline in motility and viability, our results showed that the plasma membrane surrounding the sperm head was the most vulnerable structure to cryo-damage with various degrees of swelling, undulation, or loss affecting about 50% of the total sperm population after equilibration and freezing. Typical acrosome reaction was limited to 10% of the spermatozoa after freezing. We also observed increased number of mitochondria with distorted cristae (15%). Chromatin damage was significantly increased by cryopreservation as evident by TEM (9%). This was mainly due to DNA breaks as confirmed by Sperm Chromatin Structure Assay (SCSA) (8.4%) whereas the chromatin structure was less affected as evaluated microscopically by toluidine blue staining. We concluded that, using standard cryopreservation protocol, the most pronounced damage induced by cryopreservation is observed in the plasma membrane. Further improvement of cryopreservation protocols should thus be targeted at reducing plasma membrane damage. Acrosomal, mitochondrial and chromatin damage are also evident but appear to be within acceptable limits as discussed.

Changes in the Cellular Distribution of Tyrosine Phosphorylation and Its Relationship with the Acrosomal Exocytosis and Plasma Membrane Integrity during In Vitro Capacitation of Frozen/Thawed Bull Spermatozoa

“…Furthermore, at the beginning of the incubation, we found that the spermatozoa lacking the acrosome showed the same PTP distribution than those with intact acrosome, while after three hours~80% of the former ones lost the PTP in the head (Table 3, Figure 3). Our basal level of AE detected after thawing and at time 0 could be a consequence of the freezing/thawing process as it is known to cause acrosome loss [46]. Nevertheless, it is known that ejaculated semen has also a fraction of spermatozoa that have undergone AE, which could be another explanation for this initial levels of AE [47].…”

Membrane stability and mitochondrial activity of bovine sperm frozen with low‐density lipoproteins and trehalose

“…Throughout the freezing and thawing process, spermatozoa are subjected to thermal, osmotic and oxidative stresses that result in alterations in the plasma membrane, reducing their functionality and compromising the results of fertilization (Beran et al, ; Sieme, Oldenhof, & Wolkers, ). During bovine sperm cryopreservation, the plasma membrane is the most affected structure because it may suffer from severe swelling or complete loss (Khalil, El‐Harairy, Zeidan, Hassan, & Mohey‐Elsaeed, ). Bovine spermatozoa also undergo premature capacitation, where they exhibit elevated metabolic rates and an increased membrane fluidity and permeability (Cormier, Sirard, & Bailey, ).…”

Effect of vitamin E addition to frozen Simmental bull semen extender on post-thawing quality

“…Sperm abnormality is a form of physical defect mostly on the spermatozoa cell body. A primary abnormality is a form of defection on the sperm cell head and acrosome cap, while secondary abnormality is a form of defection mostly on the tail part of the sperm cell [2,19]. This research focused mainly on secondary abnormality which was mostly occurred during frozen semen production.…”

“…These results of abnormality percentage of sperm on T1 dan T2 were also still on the range of normal standard of sperm abnormality percentage. Sperm abnormality percentage must be less than 10% in orfer to get a moderate quality of frozen semen [19]. Vitamin E addition with too much low or high level would instead increase sperm abnormality percentage above normal standard.…”