Iva Getz

Effects of bovine spermatozoa preparation on embryonic development in vitro

The aim of our research was to examine the ability of density gradient preparation BoviPure® and swim up method on bull sperm separation and in vitro embryo production (IVP) systems. Frozen/thawed semen from six Simmental bulls was pooled and treated using both methods. The sperm motility, concentration, membrane activity, membrane integrity and acrosomal status were evaluated and compared before and after sperm processing using BoviPure® and swim up methods. We also evaluated and compared cleavage rates, embryo yield and quality between the methods. There were significant differences (P < 0.05) between the sperm characteristics before and after BoviPure®, but not after swim up method. However, there were significant differences for sperm results among those two mentioned methods. A total of 641 oocytes were matured and fertilized in vitro and cultured in SOFaaBSA. The percentage of cleavage (Day 2) and the percentage of hatched embryos (Day 9) were similar for both methods. However, embryo production rate (Day 7) was significantly higher using BoviPure® method (P < 0.05). Also, total cell number and embryo differential staining (inner cell mass and trophectoderm cells) of Day 7 morulas and blastocysts showed that BoviPure® treated sperm displayed higher quality embryos compared to swim up method (P < 0.05). Our results indicate that BoviPure® method has an enhanced capacity in sperm selection for in vitro embryo production when compared with swim up method. So, we concluded that BoviPure® could be considered as a better alternative to swim up method for separating bull spermatozoa from frozen/thawed semen for IVP of bovine embryos.

Concentrations of total proteins and albumins, and AST, AP, CK and GGT activities in the blood serum Boer and Saanen goats during puerperium.

The metabolism of proteins in the blood serum in Boer and Saanen goats was investigated during puerperium. Twenty Boer goats (10 primiparous and 10 pluriparous) and 10 Saanen goats (five primiparous and five pluriparous) between 2 and 5 years of age were used in this research. Blood for analysis was taken every fourth day from day 3 until day 40 post-partum. Blood samples were collected by jugular puncture. In the obtained blood serum, the concentration of total proteins (PT) and albumin (ALB), and the activity of enzymes aspartate aminotransferase (AST) [the Enzyme Commission number (EC number) 2. 6. 1. 1.], gamma-glutamyltransferase (GGT) (EC 2. 3. 2. 2.), creatine kinase (CK) (EC 2. 7. 3. 2.) and alkaline phosphatase (AP) (EC 3. 1. 3. 1.) were determined by spectrophotometry. These parameters were in physiological ranges in Boer goats and in Saanen goats, without significant differences according to number of kids per doe. According to the research results of the blood serum in goats during puerperium, there were no significant differences in the concentration of ALB. Boer goats had significant higher (p < 0.05) concentration of PT and enzyme activity of AP, CK and GGT. Saanen goats had only enzyme activity of AST significantly higher (p < 0.05). Enzyme activity of alkaline phosphatase was significant higher (p < 0.05) in pluriparous goats in both breeds than in primiparous. The obtained results may represent a contribution to a better understanding of protein metabolism during puerperium in dairy and meat goats and for diagnostic purposes.

Optimization of Sperm for In vitro Production of Bovine Embryos

The use of in vitro technique with domestic animal gametes and the methods of embryo manipulation of assisted reproduction is currently the most advanced method in cattle breeding with regard to scientific research and practice. The technology of In vitro Production (IVP) of bovine embryos comprises In vitro maturation, In vitro Fertilization (IVF) and In vitro culture until the stage of the blastocyst. During mating, cervical mucus poses a barrier that only allows the migration of spermatozoa with normal morphology, progressive motility, and highly stable nucleus. Fresh ejaculates of bulls usually have more than 80% of progressively motile spermatozoa and 85% with morphologically normal shape. Frozen- thawed bull sperm has a lower percentage of progressive motility (30%-70%) so special attention should be paid during optimization to the methods of sperm preparation for assisted reproduction such as IVP. With the use of such procedures, the sperm quality could be significantly improved by enhancing progressive motility and increasing the number of morphologically normal spermatozoa. Such selection of spermatozoa separates motile sperm from non-motile, removes seminal plasma, cryoprotective agents, other background materials and debris, and also at the same time initiates sperm capacitation. The efficacy of sperm preparation methods could be evaluated by using different sperm parameters such as: sperm motility, morphology, concentration, viability, membrane activity, acrosome status, reactive oxygen species formation, chromatin maturity and integrity, protamination degree and IVP rates. The aim of the current review is to consider and discuss scientific data regarding the necessity of bull sperm optimization for an IVF procedure in order to improve IVP efficiency of bovine embryos. It could be concluded that the use of different sperm optimization methods for IVF is essential in order to improve the quality of obtained sperm. However, it would be advisable to extend the comparison of sperm preparation methods by transfer of IVP embryos into recipient cows which will allow more reliable results of subsequent embryo development.