Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization.

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Heterologous IVF, a replacement for homologous IVF, could provide a means to test the sperm fertility potential; to study gamete physiology and early embryo development; and to avoid the use of valuable dolphin oocytes, which are difficult to obtain. Here, we present protocols that have been successfully used to collect and cryopreserve dolphin spermatozoa. The collection of semen is performed by manual stimulation on trained dolphins. Cryopreservation is accomplished using a TRIS egg-yolk based extender with glycerol. In addition, we present a protocol that describes heterologous IVF using dolphin spermatozoa and bovine oocytes and that verifies the hybrid nature of the resulting embryo using PCR. Heterologous fertilization raises questions on fertilization and can be used as a tool to study gamete physiology and early embryo development. In addition, the success of heterologous IVF demonstrates the potential of this technique to test dolphin sperm fertilizing capacity, which is worth further examination.

Cryopreservation of canine sperm using egg yolk and soy bean based extenders.

Animal protein-based extenders are widely used despite being a potential source of bacterial or mycoplasma contamination. Its replacement with vegetal protein-based extenders could represent an interesting alternative for dog sperm cryopreservation. This technique could be further improved by the addition of Tris-Glucose-Citric acid (TGC) that could physically protect the spermatozoa and improve its homeostasis. The aim of this study was to evaluate a cryopreservation protocol for dog spermatozoa using a soybean-based extender (LP1℗) as well as the effects of the addition of (TGC) immediately after the semen collection. Eleven ejaculates from purebred adult dogs were collected, centrifuged in the absence or presence of TGC and processed as fresh or cryopreserved spermatozoa with: egg yolk-based extender (CaniPRO) or LP1℗. Freezing the spermatozoa in LP1℗ reduced the amplitude of the lateral head displacement, the percentage of spermatozoa that showed the intact acrosome and the mitochondrial function (P<0.05). These samples also showed a trend towards increased percentage of apoptotic spermatozoa (P<0.05). The addition of TGC before centrifugation did not improve the seminal parameters and adversely affected motility (P<0.05) in the spermatozoa cryopreserved in CaniPRO. However, TGC did not affect motility and increased (P<0.05) the percentage of intact acrosomes in the spermatozoa cryopreserved in LP1℗, reaching similar values than those cryopreserved in CaniPRO. In conclusion, LP1® plus TGC provide the same level of protection to dog spermatozoa cryopreservation than the egg yolk based extender CaniPRO when comparing standard post-thaw sperm quality parameters.

Effects of post-thaw incubation on motility, acrosomal integrity and in vitro fertilizing capacity of boar spermatozoa cryopreserved in 0.5 and 5ml straws.

The effect of post-thaw incubation (0 vs. 5h at 15°C) and straw size (5 vs. 0.5ml) on motility, acrosomal integrity and in vitro fertilizing (IVF) capacity of cryopreserved boar spermatozoa was studied. In samples assessed immediately after thawing, no differences were found between the two straw sizes. After 5h post-thaw incubation, all parameters, except polyspermy, decreased and, spermatozoa packaged in 5ml straws showed better functional and IVF parameters than these in 0.5ml straws.

Relationship between non-return rate and chromatin condensation of deep frozen bull spermatozoa.

This study analyzes the relationship between chromatin condensation and field fertility, expressed as 90 days non-return rate (NRR), of bulls actively used by AI studs. Frozen-thawed semen from five bulls (six ejaculates per bull, three straws per ejaculate), that showed a non-return rate between 60 and 80%, were analyzed to assess sperm chromatin condensation and stability. The chromatin condensation was determined by flow cytometry using propidium iodide as fluorochrome, and the chromatin stability was evaluated by inducing its decondensation with SDS and EDTA. Coefficient of variation among replicates was less than 7% and 5% for chromatin condensation and stability, respectively. No correlation was present between chromatin condensation and NRR. However, significant correlation was found between chromatin stability and NRR. Chromatin stability was higher (P < 0.05) in those bulls that showed higher fertility. The results obtained in this study conclude that assessment of stability could be a valuable tool for routine evaluation and identification of ejaculates with high levels of sperm chromatin abnormalities and to detect animals of higher reproductive potential.