Veterinary Research Forum

Abstract The effects of substituting whole egg yolk (WEY) in a tris-citrate-based extender with two derived fractions, including a buffer-soluble fraction (BSF) and an ammonium sulfate insoluble yolk fraction, on the freezability of bull sperm were investigated. The BSF and ammonium sulfate insoluble yolk fraction levels of egg yolk in the respective diluents were consistent with their extracted values from an equivalent volume (20.00%) of egg yolk in the control diluent. The extenders were then enriched with 0.00, 5.00, and 10.00% (v/v) of pasteurized skim milk. Semen samples were collected weekly over five consecutive weeks from six adult bulls, and the standard ejaculates were pooled. The pooled semen was subsequently divided into seven experimental extenders and frozen in 0.50 mL French straws. Various sperm quality parameters, including kinematics, acrosome integrity, capacitation, and DNA fragmentation, were evaluated post-thawing. Results indicated that sperm kinematics and the percentage of acrosome-reacted sperm in experimental extenders were not significantly different from those in the control group. The milk-free ammonium sulfate insoluble yolk fraction extender exhibited higher percentages of capacitated sperms, but lower percentages of spermatozoa with intact DNA compared to the WEY extender sperm. The addition of 10.00% milk into the BSF diluent resulted in a significant increase in the proportion of sperm with intact DNA and a notable decrease in the percentage of sperm with partially fragmented DNA compared to the control. In conclusion, a BSF extender enriched with 10.00% cow's skimmed milk is recommended as a substitute for WEY in the cryopreservation of bull semen.

Abstract Although cryopreservation of ovarian tissue has advanced greatly, it remains a challenge, and protocols should be optimized to handle the heterogeneous nature of ovarian samples. In an effort to address this factor, the present study evaluated the effects of corpus luteum (CL) and side of ovaries (right versus left) on cellular morphology and viability of vitrified bovine ovarian fragments in a closed system. The ovaries were categorized according to whether they had a CL and which side they were on, and then divided into six groups: 1) CL+ (with CL) group; 2) CL˗ (without CL) group; 3) right ovaries group; 4) left ovaries group; 5) fresh control group (ovaries without vitrification or culture that were not selected for CL or ovarian side) and 6) In vitro culture medium control group (non-vitrified ovaries that were not selected for the presence or absence of CL or side of the ovaries). The current study shows that the CL˗ and right groups had the greatest percentage of follicles with normal morphology compared to other vitrified-warmed groups. Furthermore, the levels of necrosis and tissue damage of the right cultured group were the lowest compared to other groups. It was shown that bovine ovarian tissues derived from right ovaries and ovaries without a corpus luteum can be functionally and morphologically preserved after vitrification. For the first time, the present study suggests that bovine ovarian tissue vitrification can be improved by considering the origin of the ovaries.

Abstract Sperm survives for a very short time in fresh semen, and slow cooling to 5.00 ˚C kills a large number of sperms. This study was aimed to compare the semen quality parameters and anti-oxidant levels in four extenders (manual, Triladyl, Steridyl and AndroMed). Semen samples were obtained from a total number of 12 dual-purpose Simmental bulls kept in the Simmental Cattle Breeding Center for a period of 3 months using an artificial vagina. Sperm viability, motility, abnormal morphology, plasma membrane integrity, DNA damage, chromatin quality, total antioxidant capacity (TAC) and lipid peroxidation were evaluated. The highest progressive motility, viability, plasma membrane integrity, and TAC and the lowest levels of malondi-aldehyde in the frozen-thawed semen belonged to the semen group frozen with Triladyl. Parameters of motility were higher in the frozen-thawed semen with Triladyl than in other groups, indicating a significant difference from the manual extender. Among the extenders studied, Triladyl was the most suitable for semen freezing in Simmental bulls.

Abstract Harris hawks (Parabuteo unicinctus) are the raptors with more anthropogenic use, which makes it necessary to improve their reproduction either natural or assisted for sustainable use even though they are not endangered. The aim of this study was to determine fresh and thawed P. unicinctus sperm capacitation and acrosome reaction parameters as indicators of sperm viability in ejaculates of birds with injuries. A total of 30 ejaculates were analyzed as fresh and thawed samples. Basic sperm evaluations as well as in vitro acrosomal reaction capability through the presence and distribution of Ca²⁺ and N-acetylglucosamine and sialic acid using chlortetracycline and agglutinin-fluorescein isothiocyanate Triticum vulgaris were performed,respectively. Tagged samples were observed using epifluorescence microscopy. The results indicated 30.00% decrease in sperm viability following freeze-thaw, while rate of capacitated sperms (10.00%) and the percentage of sperm with acrosomal reaction (40.00%) were increased. The results of the experiment demonstrated that the post-thaw viability of the obtained sperm may be an alternative for use in artificial insemination.

Abstract Canine seminal plasma contains antioxidant enzymes to protect sperm against internally generated ROS. These enzymes are removed from seminal plasma during the process of cryopreservation. The freezing/thawing process can cause some morphological and functional changes via ice crystallization and osmolality imbalance. The present study was conducted to evaluate the effects of curcumin supplementation on sperm total count, motility, progressive motility, viability, morphology, total antioxidant capacity (TAC), DNA integrityand NOX5 gene expression of dog frozen semen. The pooled semen was allocated to fresh (Group 1) and frozen (Group 2) controls, curcumin (2.50 mM) (Group 3) and curcumin (5.00 mM), (Group 4). Sperm parameters including total sperm count, morphology, motility, progressive motility, sperm concentration and DNA integrity in addition to TAC were evaluated in fresh and frozen-thawed semen samples. Real-time PCR was used to investigate NOX5 and GADPH (reference gene) genes expressions. Curcumin at 2.50 mM provided a greater protective effect on the DNA integrity compared to 5.00 mM and control groups. TAC was significantly higher in 2.50 mM group than other groups. NOX5 gene expression in curcumin 2.50 mM was higher than 5.00 mM group. In conclusion, curcumin seems to emolliate sperm parameters and to protect sperm against sperm reactive oxygen stress and increases NOX5 gene expression.

Abstract Previous studies have reported many discrepancies about the best type and concentration of cryoprotective agents (CPAs) and biological variability among various pre-antral follicle classes after cryopreservation of ovarian tissue. The aim of this study was to investigate the impacts of some synthetic polymers on histological characteristics of different types of pre-antral follicles after bovine ovarian tissue vitrification. From each bovine ovarian pair, fragments were recovered and immediately fixed for analysis (fresh control group) or submitted to vitrification (sucrose, X-1000, Z-1000 and polyvinylpyrrolidone groups), either followed by in vitro culture for 1 or 5 days. In this case, although, the addition of X-1000 resulted in greater percentages of normal follicles for almost all pre-antral follicle classes compared to those of other groups, there are some exceptions. These results indicate that the inclusion of polyvinylpyrrolidone in the freezing media can improve the morphology of the post-warmed transitional follicles and cultured primordial follicles on day five more than other CPAs. According to the results of this study, it can be concluded that although ovarian tissue cryopreservation is often performed to preserve the primordial follicles, by choosing the best combination of permeating and non-permeating CPAs (synthetic polymers), more advanced stages of bovine pre-antral follicles, transitional, primary and secondary follicles, may also survive the cryopreservation process.

Abstract The objective of this study was to evaluate quality of frozen-thawed bull semen processed with extenders containing vitamin C and E as antioxidants. Pooled semen's were collected from 5 bulls and diluted to a concentration of 30 × 106 sperm/mL with citrate –egg yolk (CEY) or Tris – egg yolk (TEY) extenders. The diluted semen was divided to 5 aliquots including control and 4 experimental groups. Each aliquot was further diluted with an equal volume of CEY or a Tris (hydroxymethyl) aminomethane (TRIS) - based extender without (control) or containing vitamin C 1mM or 2mM and vitamin E 0.1mM or 0.2mM, and routine semen evaluations like sperm motility, viability and measurement of lipid peroxidation (LPO) were conducted. Significant reductions of LPO were achieved by addition of 1mM vitamin C and 0.1 mM vitamin E to CEY extender (P < 0.05). The highest sperm viability was achieved by addition of 0.1mM vitamin E to CEY extender. Supplementing CEY extender with 2mM vitamin C and 0.1mM vitamin E improved the sperm motility compared with the control group. On the basis of the present results it is concluded that vitamin C and E are very efficient antioxidants in CEY extender.