Effect of different carboxylated poly l-lysine and dimethyl sulfoxide combinations on post thaw rabbit sperm functionality and fertility.

Antifreeze proteins are biologically active substances which protect living organisms against freezing injuries. The effect of a synthetic antifreeze protein carboxylated poly l-lysine (CPLL) in the extender was evaluated in the presence of a conventional cryoprotective agent, dimethyl sulfoxide (Me2SO), for freezing rabbit sperm cells. The experiment was conducted according to 2 × 3 factorial design including two Me2SO (5 or 8%) and three CPLL (0, 0.5 or 1%) concentrations. CPLL supplementation improved post-thaw live and live-acrosome intact sperm rates (P<0.01) without a prominent influence on the motility (P>0.05) and live-membrane intact (P>0.05) sperm rates. The most striking effect of CPLL supplementation was seen on the DNA integrity where it reduced DNA fragmentation of sperm cells significantly by interacting Me2SO (P < 0.01) during freezing and thawing. However, it could not replace Me2SO in the extender and did not improve pregnancy rate. In conclusion, CPLL supplementation to the extender in the presence of Me2SO improved sperm quality parameters and post-thaw DNA integrity.

Comparative efficiency of novel laparoscopic and routine vaginal inseminations with cryopreserved semen in rabbits.

Laparoscopic artificial insemination technique (LAI) is described to overcome reduced fertility problems in sheep artificial insemination (AI) programmes with frozen semen. Later on, this technology was modified for endangered non-domestic cats to deposit low quality or reduced number of sperm cells hardly obtained by electro-ejaculation into the oviduct. This technique by passes the complex structure of cervix and efficiently transfers the sperm cells to the point of fertilization. In recent years, rabbits are becoming popular transgenic animal models producing various therapeutic and commercial products, as well as being experimental animals for disease models. The worldwide transportation of frozen semen and re-establishment of transgenic lines using AI technology has become a common practice. Therefore, this study was designed to describe a laparoscopic intrauterine insemination technique, which might assist in conceiving the animals with limited number of sperm cells. The female rabbits were laparoscopically (n = 22) or vaginally (n = 13) inseminated with frozen-thawed semen samples containing approximately 10 × 106 motile sperm. The laparoscopic insemination technique provided higher pregnancy rate (45.5%) than vaginal insemination technique (7.7%) (p < .05). In conclusion, the described laparoscopic AI might be a new alternative technique, thus enabling limited or low-quality frozen sperm samples to establish pregnancy in rabbits.

Effect of oviductal fluid on bull sperm functionality and fertility under non-capacitating and capacitating incubation conditions.

This study investigated the effect of bovine oviductal fluid from late follicular (LF) and early luteal (EL) phases on bull sperm functionality under non-capacitating (NCAP) and capacitating (CAP) conditions. Frozen-thawed semen samples from five bulls were thawed and incubated (0, 1 or 2 h) in NCAP and CAP media supplemented with 1% bovine oviductal fluid (LF and EL groups) and in absence of fluid (C group). Motion parameters were assessed by CASA; sperm viability, acrosomal integrity and membrane lipid disorder parameters were evaluated by flow cytometry; and sperm DNA fragmentation was evaluated by the Comet assay. Finally, in vitro fertilization with sperm treated under CAP conditions was performed and further embryo culture results evaluated. In NCAP medium, addition of LF and EL fluid increased the total and progressive motility, and LF fluid improved the stability of sperm DNA. However, under CAP conditions addition of LF and EL fluid decreased some sperm motion parameters and some parameters of sperm DNA stability. Proportion of viable sperm cells with low lipid disorder was higher in NCAP than CAP medium and addition of LF fluid markedly increased the proportion of viable spermatozoa with high lipid disorder and acrosome alteration (spontaneous acrosome reaction). Under current conditions, incubation of bull sperm with oviductal fluid before insemination did not affect detrimentally the IVF results nor embryo development, being blastocyst rate similar between CAP-LF, CAP-EL and control groups. In conclusion, oviductal fluid positively influences sperm functionality and modulate in vitro capacitation.

Sperm DNA and detection of DNA fragmentations in sperm.

The questionable effectiveness of routine sperm parameters in determining male factor infertility problems and increasing the success rates of assisted reproductive techniques have led to the investigation of more detailed sperm parameters that could affect the male fertility and reproduction. Thus, the effects of different sperm parameters such as sperm DNA integrity was started to be investigated thanks to the previously described methods such as single cell gel electrophoresis (COMET) assay, sperm chromatin structure assay (SCSA), acridine orange test (AOT), terminal deoxynucleotidyl transferase-mediated deoxyuridine (TdT) triphosphate (dUTP) nick end labeling (TUNEL) assay and sperm chromatin dispersion (SCD) test. However, studying on sperm DNA might be very complex because the sperm DNA differs from the somatic cell DNA with its unique structure. Also, the sperm DNA undergoes many changes during spermatogenesis and it is condensed by being packaged tightly with different types and numbers of protamines in different species. Despite all these difficulties, these methods provide important information about the reasons and consequences of DNA damages in sperm and the effects of these damages on reproduction.

Protective effect of cholesterol-loaded cyclodextrin pretreatment against hydrogen peroxide induced oxidative damage in ram sperm.

Three experiments were conducted to determine the protective effect of cholesterol-loaded cyclodextrin (CLC) against hydrogen peroxide (H2O2) or cryo-induced damage in ram sperm. In Experiment 1, the fresh ejaculates were either treated with CLC or remained untreated. Both CLC treated and untreated samples were then incubated with 0, 250 or 500 µM H2O2 at 35°C for 12 h. After incubation period of 12 h, the motility, viability and membrane integrity remained higher in CLC treated sperm even in the presence of 250 or 500 µM H2O2. The H2O2 treatment affected all the sperm parameters adversely (P<0.05). However, compared to CLC untreated counterpart, the motility, viability and membrane integrity remained higher (P<0.05) in treated sperm, even in the presence of 250 or 500 µM H2O2 during 12 h of incubation. In Experiment 2, semen was cryopreserved in the presence or absence of CLC. The post-thaw results revealed that CLC treated sperm has higher (P<0.05) motility, viability and membrane integrity compared to the control. In Experiment 3, lipid peroxidation levels were assessed by determining malondialdehyde (MDA) concentrations during the H2O2-induced oxidative stress in CLC treated and untreated sperm. However, no difference (P>0.05) in MDA level was observed among the groups at any stage of incubation. In conclusion, the CLC incorporation in ram sperm membrane may protects it against H2O2 or cryo-induced oxidative damage. The cryoprotective influence of CLC on ram sperm might be resulted from, at least partly, its antioxidative property.

Comparison of two different cryopreservation protocols for freezing goat semen.

In this study, two different semen cryopreservation protocols were compared to freeze goat semen. The ejaculates (n=12) were collected by using electro-ejaculator from six mature bucks (two ejaculates per each buck). Each ejaculate was divided into two groups as Protocol 1 (P1) and Protocol 2 (P2). In P1, semen was diluted directly in an extender containing 15% egg yolk, 300mM Tris, 28mM glucose, 95mM citric acid 5% glycerol to a concentration of 200×10(6)sperm/mL. In P2, after the removal of seminal plasma by centrifugation, the semen sample was diluted with the first portion of milk extender consist of 100mg/mL skimmed milk powder and 27.75mM glucose (without glycerol) to a concentration of 400×10(6)sperm/mL. The second portion of the milk extender containing 14% glycerol was added to semen gradually in order to achieve sperm concentration 200×10(6)sperm/mL and 7% glycerol level in the final volume. Extended semen was loaded in 0.25mL straws, held for 2h at 4°C, frozen in nitrogen vapor and stored in liquid nitrogen. Post-thaw motility and live sperm rate (mean±SEM) were significantly lower (P<0.05) in P1 as compared to P2 (47.50±1.23% vs. 55.63±1.72%; 80.04±1.29% vs. 84.04±1.08%, respectively). However, live intact, total intact, abnormal, reacted acrosome and DNA damaged sperm rates were similar (P>0.05) in both protocols. It was concluded that both protocols used in this study provided reasonable post-thaw parameters; however, P2 yielded better motility and live sperm rate compared to P1.