Addition of orange, pineapple and beet juices as extenders for cryopreservation of ram semen

Searching for improvements in semen cryopreservation, natural substances are commonly studied focusing to improve the sperm quality. The aim of this study were evaluated the effect of adding orange, pineapple, and beet juices in different concentrations and combinations to the ram semen cryopreservation extender. Five ejaculates from five adult rams were used. The semen pool was diluted in egg yolk-based extender and mixed with the following 15 treatments (at a final concentration of 400.10⁶ sptz/mL): orange 10% (O10) and 15% (O15); pineapple 10% (P10) and 15% (P15); beet 10% (B10) and 15% (B15); pineapple + orange 10% (PO10) and 15% (PO15); pineapple + beet 10% (PB10) and 15% (PB15); beet + orange 10% (BO10) and 15% (BO15); pineapple + beet + orange 10% (PBO10) and 15% (PBO15); and the control group (CON). Post-thaw in 0.25 mL straws semen quality analysis of cryopreserved semen was performed by CASA and flow cytometry. Analysis of variance (PROC GLM) was carried out and the averages were compared using the SNK test. Pearson's correlation test was also performed. No effect was noted in the addition of juices to the semen extender prior to cryopreservation. Post-thawed, although, statistically similar to the control group, the total motility of the B10 group reached acceptable standards of total motility. In addition, B10 group showed the highest values (p<0.05) of progressive motility than control group or other treatments. The addition of 10% beet juice to the ram semen extender can improve the cryopreservation of sperm motility. Keywords: antioxidant; freezing; sperm; semen extender

Addition effect of pineapple, orange and beet juices on the mitochondrial membrane potential of ram sperm

Sperm cells are specialized for the transport of gametes, requiring a great amount of energy to move about, and mitochondrial membrane potential is an important parameter when evaluating mitochondrial activity in a cell. Plants have thousands of phytochemical compounds in their roots, leaves, fruits, flowers and stems, which have several biological effects, such as anti-oxidant, anti-inflammatory, anti-carcinogenic and anti-inflammatory action. The objective of this work was to evaluate the effects of adding pineapple, beetroot, and orange juices individually and in different combinations at concentration of 10% in the cryopreserved semen extender of rams, through the mitochondrial membrane potential of sperm cells. At the Institute of Zootechnics in Nova Odessa, SP, five semen collections from five rams were performed during a two-week period with the use of an artificial vagina. After collection, the semen was analyzed with a microscope following the recommendations and standards of sperm kinetics according to the CBRA. On each day, after the collection and analysis of the semen of all rams, a pool was formed to minimize the influence of individual traits of the animals. The sperm concentration was corrected to 800.10ⶠsptz / ml with addition of the BotuBov® semen extender and subsequently the juices, after the fruits and beetroots were washed with distilled water, peeled, cut into pieces and squeezed in nylon mesh, were added to the semen extender, obtaining the treatments: pineapple (P), beetroot (B), orange (O), pineapple and orange (P + O), pineapple and beetroot (P + B), beetroot and orange (B + O) and pineapple, beetroot and orange (P + B + O), at a concentration of 10 %, and the control group. The samples were packed in 0.25 ml straws with a concentration of 100.10ⶠsptz per straw, submitted to cryopreservation with a negative curve of -0.25 °C and stored in liquid nitrogen for further analysis. The analyses were performed at USP Pirassununga and the Accuri® flow cytometry system was used to measure low mitochondrial membrane potential (LMP) and high mitochondrial membrane potential (HMP) through a JC-1 fluorescence probe. In the LMP parameter, treatments did not differ (p>0.05), with values of 27.31% ± 19.36, 21.62% ± 10.55, 43.43% ± 21.6, 31,72% ± 29.23, 19.73% ± 5.05, 31.34% ± 24.37, 33.67% ± 23.94 and 21.78% ± 7.25 for control group, P, B, O, P + B, P + O, B + O and P + B + O respectively. In the HMP parameter, the P + B + O treatment caused the highest percentage of high mitochondrial membrane potential, with a value of 21.15% ± 9.89, greater (p0.05). Interestingly, the P + B + O treatment did not differ (p>0.05) from treatments P, P + B and P + L, with values of 14.73% ± 6.19, 17.94% ± 9.32 and 14.74% ± 4.56, respectively, which did not present differences (p>0.05) from the other treatments, except for B, which was lower than P + B. The addition of pineapple juice in the different treatments culminated in a gain in the percentage of sperm with high mitochondrial membrane potential, showing a possible agonist effect of compounds present in this fruit

Addition effect of pineapple and beetroot juices on the motility parameters of ram semen extender for cryopreservation

Sperm motility is measured by the number of live sperm cells with progressive or non-progressive movement, which allows them to travel to the isthmus of the uterine tube where fertilization occurs. Plants such as pineapple and beetroot are rich in phytochemical compounds that can have positive effect on cellular metabolism, in addition to making the extracellular environment more suitable for the maintenance of cell function. The objective of this work was to evaluate the effects of the addition of pineapple and beetroot juice at 10 and 15% concentrations on the parameters of total motility and progressive motility in cryopreserved sheep semen. At the Institute of Zootechnics in Nova Odessa, SP, five semen collections from five rams were performed during a two-week period with the use of an artificial vagina. After collection, the semen was analyzed under a microscope following the recommendations and standards of sperm kinetics according to the CBRA. On each day, after the collection and analysis of the semen of all rams, a pool was formed to minimize influence of the individual traits of the animals. The sperm concentration was corrected to 800.10ⶠsptz / ml with addition of the BotuBov® semen extender and subsequently the juices were added separately for each treatment. The pineapples and beetroots were washed with distilled water, peeled, cut into pieces and squeezed in nylon mesh to obtain the juices, which were added to the semen extender,  obtaining the treatments: pineapple 10 and 15% and beetroot 10 and 15%, in addition to the control group. The samples were packed in 0.25 ml straws with a concentration of 100.10ⶠsptz per straw, submitted to cryopreservation with a negative curve of -0.25 °C and stored in liquid nitrogen for further analysis. Analyses were performed at USP Pirassununga and computer-assisted sperm analysis (CASA) was used to measure the kinetic parameters of the semen. In the total motility parameter the control group, 10% beetroot and 15% beetroot were similar (p>0.05) with values of 23.2% ± 6.1, 31.5% ± 11.4 and 22.4% ± 9.5 respectively, whereas treatments with 10 and 15% pineapple juice were lower than the others (p 0.05). In the case of progressive motility, the treatment with beetroot at 10% was superior to the others (p 0.05), with values of 3.1% ± 2.3, 1.6% ± 1.1 and 7.6% ± 3.6, respectively. The addition of 10% and 15% beetroot juice to the BotuBov® semen extender maintained acceptable levels of total motility and increased the number of cells in progressive movement, indicating it can be an alternative product of natural origin for the formulation of semen extenders for rams