Recently, we generated transposon-transgenic boars (Sus scrofa), which carry three monomeric copies of a fluorophore marker gene. Amazingly, a ubiquitous fluorophore expression in somatic, as well as in germ cells was found. Here, we characterized the prominent fluorophore load in mature spermatozoa of these animals. Sperm samples were analyzed for general fertility parameters, sorted according to X and Y chromosome-bearing sperm fractions, assessed for potential detrimental effects of the reporter, and used for inseminations into estrous sows. Independent of their genotype, all spermatozoa were uniformly fluorescent with a subcellular compartmentalization of the fluorophore protein in postacrosomal sheath, mid piece and tail. Transmission of the fluorophore protein to fertilized oocytes was shown by confocal microscopic analysis of zygotes. The monomeric copies of the transgene segregated during meiosis, rendering a certain fraction of the spermatozoa non-transgenic (about 10% based on analysis of 74 F1 offspring). The genotype-independent transmission of the fluorophore protein by spermatozoa to oocytes represents a non-genetic contribution to the mammalian embryo.
The aim of the present study was the modification and evaluation of three different semen extenders for cockatiel semen in order to achieve a long survival time for transport, examination purpose and for potential cryopreservation, respectively. Therefore, individual and pooled semen samples of 30 cockatiels (Nymphicus hollandicus) were investigated for pH and osmolality values and subsequently pH and osmolality values of the semen extenders were adjusted to those values in the semen. Pooled semen samples were then partitioned into four equal parts and diluted with the three different semen extenders in 1:4 and 1:8 dilution. 1 % glucose-Ringer’s solution was used as control, respectively. A total of 64 incremental diluted semen samples were obtained for investigation. Each dilution was investigated regarding sperm motility immediately after dilution and another four times every 30 minutes. Sperm viability was evaluated 0 and 120 minutes after dilution via eosin B-stain on the diluted semen samples and in pure semen samples. Additionally, the fluorescence stain SYBR® Green/propidium iodide was used to assess sperm viability. The results indicate that cockatiel spermatozoa are highly sensitive to variations in pH and osmolality, requiring adjustment of commercial diluents to pH = 7.42 and osmolality = 300 mOsm/kg. Modified Lake diluent maintained higher viability and motility than other diluents tested. Sperm morphology was indicated to be least adversely affected by modified Lake diluent in 1:4 concentration compared to other semen extenders and concentrations used.
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