Effect of Honey, Coenzyme Q10, and β-Carotene/α-Tocopherol as Novel Additives in Rabbit-Sperm Cryopreservation Extender

Abstract

The effectiveness of rabbit-sperm cryopreservation is still below average compared to other domestic species. After the sperm cryopreservation process, post-thawing parameters like motility and membrane integrity are significantly compromised. The use of new extender constituents is an approach that can be used to improve the effectiveness of cryopreservation. Accordingly, we used honey (1.25, 2.5, 5, and 10%), coenzyme Q10 (100 and 200 μM), and β-carotene/α-tocopherol (500 μM/620 μM and 250 μM/310 μM) as candidate components for rabbit-sperm extenders during cryopreservation. Ejaculates from commercial adult rabbit bucks (n = 5) were cryopreserved using conventional freezing. Several post-thawing sperm parameters were assessed, including total motility, membrane integrity, viability, nuclear membrane integrity, acrosome reaction, and mitochondrial membrane potential and activation. Additionally, we performed hormonal analyses of the seminal plasma. Moreover, we analyzed the post-thawing levels of a molecular marker of sperm quality, proAKAP4, which was used in rabbits for the first time. Our findings showed that the 2.5% honey supplementation increased the post-thawing sperm motility (13.75 ± 3.75%) compared to the greater concentrations employed. However, the post-thawing motility was negatively affected by the coenzyme Q10 (0%, in both groups) but was not affected by the β-carotene/α-tocopherol supplementation (22 ± 18.15%, and 11.67 ± 10.17%). In conclusion, the cryopreservation protocols of this study did not help to maintain the sperm parameters after thawing. Further studies are required to identify novel protocols to mitigate the damage caused to rabbit sperm during cryopreservation.

ProAKAP4 as Indicator of Long-Lasting Motility Marker in Post-Thaw Conditions in Stallions

“…However, analyzing the descriptors of the sperm trajectory only the velocity parameters (VCL, VCL and VAP) and the lateral head displacement (ALH), and only at 1 h of post-thaw incubation but not later, correlate positively with proAKAP4 concentration. This correlation has been previously found in stallions [13] and in bulls [20,26,27,35]. In other mammals, like bulls, progressive motility [39] and velocity descriptors [41,42] have been described as sperm quality and even fertility markers.…”

“…ProAKAP4 is the precursor of A-kinase anchor protein 4, which is the main protein of the fibrous sheath of the principal piece of the flagellum [12,18]. As an important part of the flagellum [19,20], proAKAP4 is a good marker of the postmeiotic stage of spermatogenesis [12,21,22] as well as a good marker of sperm progressive and total motility in stallions [13][14][15][16] and in other mammals like bulls [23], jackass [24], dogs [25], rabbits [26], rams [27] and dromedary [28]. In stallions, proAKAP4 has been established as a good marker of motility descriptors [13][14][15][16][17], since more deeper, rapid spermatozoa show a greater concentration of proAKAP4 [14] and ejaculates with a high percentage of slow sperm subpopulations show a lower concentration of proAKAP4 [16].…”

Evaluation of a commercial proAKAP4 kit for the assessment of fresh and frozen–thawed feline spermatozoa

“…For example, in horses, the first reported values for frozen-thawed semen were between 3 and 12 ng/mL (isolated from around 7.5 × 10 6 spermatozoa), while 2 years later, the same team in another study obtained a mean concentration 60.9 ± 2.1 ng/10 million spermatozoa (Blommaert et al, 2019(Blommaert et al, , 2021 TA B L E 5 Semen parameters in frozenthawed samples (n = 22) and Spearman's rank correlation coefficient (r 2 ) between proAKAP4 concentration in fresh (Fr, n = 17) or frozen-thawed (FT, n = 22) samples and sperm motility parameters post-thaw. (Riesco et al, 2020), and around 300 ng/mL per million spermatozoa in rabbits (Gardela et al, 2023). This could reflect species and individual variation, but also differences in kit reagents, as reported in this study.…”

“…For example, in horses, an extender with mannitol better preserved sperm proAKAP4 content (and motility parameters) than one with glycerol (Blommaert et al, 2021), whereas in pigs, there was a higher post-thaw proAKPA4 concentration in samples supplemented with Holothuroidea extract (Kowalczyk et al, 2022). In rabbit spermatozoa frozen in different extenders, the proAKAP4 concentration dropped to zero for some extenders, but was more than 160 ng/ mL for others (Gardela et al, 2023). As there was no correlation between proAKAP4 values in fresh semen and post-thaw quality in our study, this marker cannot be used to predict sperm freezability in domestic cats.…”

“…during cryopreservation. Regarding the latter, proAKAP4 has been used successfully to compare different cryoprotectants (Blommaert et al, 2021) or freezing extender supplements (Gardela et al, 2023;Kowalczyk et al, 2022).…”