Determination of Sex Ratio In Bovine Semen Using SYBR Green Real-Time PCR

A SYBR green real-time PCR assay was developed to find out the sex skewness in bovine sex-sorted semen samples. The qPCR assay of PLP and SRY genes revealed the mean values of X- and Y-bearing spermatozoa as 50.24 ± 0.65 and 49.75 ± 0.62 per cent in unsorted, and 91.80 ± 0.79 and 8.20 ± 0.73 per cent in X-enriched semen samples respectively.. The amplification efficiencies of the PLP and SRY primers were 99.25 and 98.03 per cent respectively. The method was validated by a series of repeatability and reproducibility assays which revealed low co-efficients of variations as 2.19 and 3.12 per cent respectively Thus becoming a reliable and inexpensive tool to evaluate the sorted semen on routine basis and validation of other sperm sexing technologies.

Staphylococcus-Induced Bacteriospermia In Vitro: Consequences on the Bovine Spermatozoa Quality, Extracellular Calcium and Magnesium Content

Bacterial contamination of bovine ejaculates intended for artificial insemination may be reflected in a significant economic loss due to unsuccessful fertilization as well as health issues of the recipients. The Staphylococcus genus represents a large part of bacteriocenosis of bovine ejaculates. Therefore, this study aims to get a closer look on the effects of Staphylococcus-induced bacteriospermia under in vitro conditions on bovine sperm quality. Prior to inducing bacteriospermia, spermatozoa were separated from each ejaculate using Percoll® Plus gradient medium in order to limit the effects only to the selected bacterial species. Seven Staphylococcus species previously isolated from bovine semen were used for our experiments at a turbidity of 0.5 McFarland (equivalent to 1.5 × 108 colony-forming units per mL). The contaminated semen samples were incubated at 37 °C and at times of 0, 2, and 4 h, motility, mitochondrial membrane potential, reactive oxygen species (ROS) generation, sperm DNA fragmentation, and magnesium (Mg) and calcium (Ca) extracellular concentration were analyzed and compared with the control group (uncontaminated). The results showed no significant changes at the initial measurement. However, significant adverse effects were observed after 2 h and 4 h of incubation. Most notably, the presence of S. aureus, S. warneri, S. kloosii, and S. cohnii caused a significantly increased ROS production, leading to sperm DNA fragmentation, changes in the mitochondrial membrane potential, and a decreased sperm motility. Furthermore, the presence of Staphylococcus species led to lower extracellular concentrations of Mg and Ca. In conclusion, the overgrowth of Staphylococcus bacteria in bovine semen may contribute to oxidative stress resulting in sperm DNA fragmentation, altered mitochondrial membrane potential, and diminished sperm motility.

Effect of extender supplemented with date palm pollen grain on bovine semen qualities

Natural extract from plant-based has grown in popularity as protective properties in extender for preserving animal semen. Date palm pollen grain (DPPG) is commercially used for male fertility by enhancing sperm count, motility and DNA quality because DPPG contains flavonoids. Thus, the present study aimed to evaluate the effect of a) extender supplementation with different concentration of DPPG on sperm motility, viability and membrane integrity b) different preservation storage of chilled and frozen bovine semen after seven days. The semen was collected through electrical stimulation and assigned to four treatment groups. The semen were diluted in Tris citric fructose egg yolk (TCFY) diluent (control group; CG) or supplemented with DPPG with different concentration (G1 = 2%, G2 = 4% and G3 = 6% in 20 mg DPPG/40 mL of Tris citric fructose (TCF). Semen samples were chilled (experiment 1) in the refrigerator (4°C) for seven days and cryopreserved in liquid nitrogen (experiment 2) prior to dilution to four treatments. The samples were thawed in a water bath (37 °C) and analysed for motility, membrane integrity and viability by conventional laboratory methods. No significant difference was observed among treatment groups in experiment 1. However, in the second experiment, the addition of 6% DPPG resulted significantly higher (p<0.05) in sperm viability compared to control groups (71.25±1.04) vs (56.47±4.69). The supplementation of 6% DPPG showed the ability to protect the viability of bovine sperm, respectively.

Efeito da adição do extrato de Jabuticaba (Myrciaria cauliflora) e Noni (Morinda citrifolia) ao sêmen bovino in natura e pós descongelado – Análise Preliminar / Effect of the addition of Jabuticaba (Myrciaria cauliflora) and Noni (Morinda citrifolia) extract to bovine semen in natura and post-thaw - preliminary analysis

No intuito de melhorar os índices de viabilidade espermática após os processos de criogenia, pesquisadores buscam desenvolver diluidores que minimizem as injurias causadas pela técnica. O objetivo do presente trabalho foi avaliar a viabilidade dos espermatozoides bovinos fresco e pós descongelado após a adição de um diluidor comercial enriquecido com extratos de plantas. Para avaliação a fresco, foram utilizados 16 ejaculados in natura obtidos de oito touros de raças Zebuínas cujo sêmen foi coletado com vagina artificial. Para análise pós descongelamento, 16 palhetas de sêmen foram obtidas a partir destes mesmos animais e congeladas por período de 30 dias, sendo descongeladas por banho maria. Os experimentos foram definidos como sêmen a fresco e pós descongelamento seguindo dos seguintes grupos experimentais: Tryladyl®, somente o diluidor; Tryladyl® Jabuticaba, utilizando extrato aquoso da casca da jabuticaba (Myrciaria cauliflora) e Tryladyl® Noni, utilizando extrato aquoso de Noni (Morinda citrifolia). Foram avaliados motilidade e vigor. Para a análise estatística foi realizado a análise de variância (ANOVA), seguida pelo comparativo de médias Tukey, ao nível de significância de 0,05%. Na análise dos dados não foi observado diferença (p0,05) entre o diluidor comercial e os grupos adicionados os extratos de plantas. Houve diferença apenas quando estes foram comparados as amostras a fresco e pós descongelado, respectivamente (p0,05). A utilização de extratos de plantas com ação antioxidantes ou ativadora são promissores para melhorar a resposta de células espermáticas.

Fertility Analysis of Bovine Semen by in Vitro Fertilization

The aim of the present study was to evaluate the efficiency of using in vitro fertilization to validate semen fertility for artificial insemination. Cryopreserved semen from ten bulls (five Nelore and five Brangus bulls) was evaluated using in vitro production of embryos (IVPE) and via fixed-time artificial insemination (FTAI). There was variation (p < 0.05) in the IVPE (20.9 to 53.7% of blastocyst production) and in the FTAI (42.0 to 56.0% of pregnant cows) results among the Nelore bulls evaluated. According to the results there was a positive correlation (rs = 0.8378; p = 0.0001) between the rate of blastocyst production (using IVPE) and the rate of pregnancy (using FTAI) using Nelore bull semen. Variation was also found between the Brangus bulls (p < 0.05), in the rates of blastocyst production (36.5 to 47.0%) and pregnancy (45.6 to 52.2%) via FTAI. There was also a positive correlation (rs = 0.8786; p = 0.0001) between the rates of blastocyst production (IVPE) and pregnancy (FTAI) when using Brangus bull semen. According to the results, IVPE may be used in addition to conventional semen analysis to evaluate and validate the semen fertility of bulls for artificial insemination programs.

Effects of Apigenin and Astragalus Polysaccharide on the Cryopreservation of Bull Semen

The purpose of this study was to determine the effects of apigenin and astragalus polysaccharides on the cryopreservation of bovine semen. Apigenin, astragalus polysaccharides, or their combination were added to a frozen diluent of bovine semen. Afterwards, Computer Assisted Semen Analysis (CASA), membrane functionality, acrosome integrity, mitochondrial integrity, CAT, SOD, GSH-Px, MDA, and ROS detection were conducted. The results showed that adding 0.2 mmol/L AP or 0.5 mg/mL APS could improve the quality of frozen sperm. Compared to 0.2 mmol/L AP alone, the combination of 0.2 mmol/L AP and 0.3 mg/mL APS significantly increased the total motility (TM), average path distance (DAP), straight line distance (DSL), average path velocity (VAP), curvilinear velocity (VCL), wobble (WOB), and sperm CAT and SOD levels (p < 0.05), while reducing the ROS and MDA levels (p < 0.05). These results indicated that the addition of 0.2 mmol/L AP or 0.5 mg/mL APS alone has a protective effect on the freezing of bovine semen. Compared to the addition of 0.2 mmol/L AP, a combination of 0.2 mmol/L AP and 0.3 mg/mL APS could further improve the quality of frozen semen.