The effect of cryopreservation media on the quality of ß-thalassemia mouse spermatozoa.

Background: The mouse model of human diseases is commonly used for biomedical study, including ß-thalassemia (ß-thal), an inherited hemoglobin disorder. Maintaining the mice strain by natural mating systems is costly and seems impractical, especially during the COVID-19 pandemic. Sperm-freezing is a cost-effective solution for ß-thal mouse colony management. Aim: To determine appropriate cryopreservation media for ß-thal mouse spermatozoa to establish a ß-thal mouse sperm bank. Methods: The epididymal spermatozoa of C57BL/6 wild-type (WT) and ß-globin gene knockout thalassemia (BKO) mice were frozen in four freezing media: I) raffinose-skim milk-monothioglycerol (MTG), II) raffinose-skim milk-glutamine, III) raffinose-egg yolk-glycerol, and IV) egg yolk-TES-Tris. The sperm quality was assessed prior to and following freeze-thawing. Results: Compared with WT counterparts, the viable spermatozoa before freezing exhibiting elevated levels of oxidative stress were significantly greater in BKO (p = 0.01). After thawing, the membrane integrity of BKO spermatozoa preserved in I was significantly lower (p = 0.001). The sperm viability and membrane integrity of BKO males were also inferior when media III and IV were used (p = 0.008-0.027). The amount of oxidative stress in the spermatozoon of BKO mice was significantly greater when preserved in I, III, and IV (p = 0.002-0.044). Comparing freezing media, the motility and acrosome integrity of WT and BKO spermatozoa preserved in IV were significantly higher than those in other media (p < 0.001 to p = 0.01). Spermatozoa with the highest mitochondrial membrane potential were observed in I in both genotypes (p = 0.012 to p > 0.05). The viability, membrane integrity, and oxidative stress of post-thaw BKO spermatozoa did not significantly differ among freezing solutions. Conclusion: Irrespective of freezing media, spermatozoa of BKO males are rather more sensitive to cryopreservation than those of WT. Raffinose-skim milk-MTG/glutamine, raffinose-egg yolk-glycerol, and egg yolk-TES-Tris can all be used to preserve BKO mouse spermatozoa. However, with slightly better sperm characteristics, egg yolk-TES-Tris may be a diluent of choice for BKO mouse sperm cryopreservation. The addition of a reducing agent to thawing media is also strongly recommended to efficiently prevent oxidative stress and therefore improve frozen-thawed sperm survival.

Influence of seasonal differences on semen quality and subsequent embryo development of Belgian Blue bulls.

Belgian Blue bulls are more susceptible to high temperature and humidity index (THI) than most other cattle breeds. Here, we investigated whether high ambient temperature during summer affected semen quality and subsequent embryo development in Belgian Blue cattle. For this purpose, semen samples were collected from six healthy mature Belgian Blue bulls in March (Low THI group; THI between 30.6 and 56.4) and August 2016 (High THI group; maximum THI of 83.7 during meiotic and spermiogenic stages of spermatogenesis; 14-28 days prior to semen collection) respectively. Motility, morphology, acrosome integrity, chromatin condensation, viability, and reactive oxygen species production were assessed for frozen-thawed semen. Moreover, the efficiency of blastocyst production from the frozen-thawed semen samples of the two groups was determined in vitro. Blastocyst quality was determined by assessing inner cell mass ratio and apoptotic cell ratio. Fresh ejaculates showed a higher sperm concentration in low THI when compared to the high THI group (P ≤ 0.05), whereas semen volume, subjective motility, and total sperm output were not affected (P > 0.05). In frozen-thawed semen, total and progressive motility, viability, and straight-line velocity were lower in high THI compared to the low THI group (P < 0.05), while H2O2 concentration, aberrant chromatin condensation, and abnormal spermatozoa were higher in the high THI group (P < 0.05). Blastocyst rates were significantly higher when low THI samples were used (P < 0.05). Moreover, the total cell number and trophectoderm cells were significantly higher (P < 0.05) in blastocysts derived from low THI samples, whereas the apoptotic cell ratio was significantly higher (P < 0.01) in blastocysts derived from high THI spermatozoa. In summary, our data show that elevated ambient temperature and humidity during summer can decrease the quality of frozen-thawed spermatozoa in Belgian Blue bulls and also affect subsequent embryo development.