Paternal effect does not affect in vitro embryo morphokinetics but modulates molecular profile.

The use of different sires influences in vitro embryo production (IVP) outcome. Paternal effects are observed from the first cleavages until after embryonic genome activation (EGA). Little is known about the mechanisms that promote in vitro fertility differences, even less about the consequences on embryo development. Therefore, this study aimed to evaluate the paternal effect at fertilization, embryo developmental kinetics, gene expression and quality from high and low in vitro fertility bulls. A retrospective analysis for bull selection was performed using the In vitro Brazil company database from 2012 to 2015. The dataset was edited employing cleavage and blastocyst rates ranking a total of 140 bulls. Subsequently, the dataset was restricted by embryo development rate (blastocyst/cleaved rate) and ten bulls were selected as high (HF; n = 5) and low (LF; n = 5) in vitro fertility groups. IVP embryos derived from high and low fertility bulls were classified according to their stage of development (2 cells, 3-4 cells, 6 cells, 8-16 cells), at 24, 36, 48, 60, 72 hpi, respectively, to evaluate embryo kinetics. Pronuclei formation (24 hpi), cleavage rate (Day 3), development rate, and blastocyst morphology (Grade I and II - Day 7) were also assessed, as well as the abundance of 96 transcripts at 8-16 cell stage and blastocysts. There was no difference in early embryo kinetics (P > 0.05), and cleavage rate (HF = 86.7%; LF = 84.9%; P = 0.25). Nevertheless, the fertilization rate was higher on HF (72%) than LF (62%) and the polyspermy rate was lower on HF compared to LF (HF:16.2% LF:29.2%). As expected, blastocyst rate (HF = 29.4%; LF = 16.0%; P < 0.0001) and development rate (HF = 33.9% LF = 18.9%; P < 0.0001) were higher in HF than LF. At the 8-16 cell stage, 22 transcripts were differentially represented (P ≤ 0.05) between the two groups. Only PGK1 and TFAM levels were higher in HF while transcripts related to stress (6/22, ∼27%), cell proliferation (6/22, ∼27%), lipid metabolism genes (5/22, ∼23%), and other cellular functions (5/22, ∼23%) were higher on LF embryos. Blastocysts had 9 differentially represented transcripts (P ≤ 0.05); being only ACSL3 and ELOV1 higher in the HF group. Lipid metabolism genes (3/9, 33%) and other cellular functions (6/9, 67%) were higher in the LF group. In conclusion, the timing of the first cleavages is not affected by in vitro bull fertility. However, low in vitro fertility bulls presented higher polyspermy rates and produced 8-16 cells embryos with higher levels of transcripts related to apoptosis and cell damage pathways compared to high in vitro fertility ones. Evidence such as polyspermy and increase in apoptotic and oxidative stress genes at the EGA stage suggest that embryo development is impaired in the LF group leading to the reduction of blastocyst rate.

Estimate of in vitro embryo production based on sperm subpopulations in Senepol bulls.

In cattle, in vitro embryo production (IVEP) is an important reproductive biotechnology responsible for the rapid expansion of the Senepol breed in our country. This breed has shown important results when used in crossbreeding and estimate IVEP in Senepol based on seminal analysis would be valuable for the semen cryopreservation industry, research institutes and breeders. Combining the evaluation of sperm subpopulations with analysis of other sperm attributes may help to improve fertility predictions in cattle. Therefore, the objectives of the present study were to: 1) identify and characterize motile sperm subpopulations in cryopreserved Senepol semen following the washing process carried out before in vitro fertilization, and 2) to determine an model for estimate IVEP based on sperm subpopulations in conjunction with other sperm quality analyzes. Samples of 38 cryopreserved semen from 28 Senepol bulls, chosen based on retrospective data from 386 IVEP routines, underwent the semen washing and were evaluated by the computer-assisted sperm analysis system. Sperm morphology was evaluated by wet preparation technique, and plasma and acrosomal membranes integrity, mitochondrial potential, oxidative status and chromatin resistance were analyzed by flow cytometry. After multivariate analysis of principal components and grouping, three sperm subpopulations were identified: SBP1 (fast and progressive motility), SBP2 (hyperactivated motility) and SBP3 (slow non-progressive motility). After categorization of IVEP in high, medium and low embryo yield, logistic regression analysis was applied to associate the results of subpopulations and other sperm quality variables with IVEP. The SBP1 and SBP2 variables affected embryo production, and an IVEP estimation model was generated for Senepol bulls based on these two subpopulations: embryo yield = 0.1563 + 0.0328 (SBP1) + 0.0173 (SBP2). SBP1 and SBP2 represents the absolute value of the percentage of subpopulations in semen. If the calculated value (by this equation) is close to 1, the embryo yield will be low; if is close to 2, will be medium; if is close to 3, will be high. In conclusion, three subpopulations were found for Senepol semen and, despite all analyzed variables, only SBP1 and SBP2 were included in the model to estimate IVEP in this breed.

Carnosine as malondialdehyde scavenger in stallion seminal plasma and its role in sperm function and oxidative status.

Semen biotechniques may impair sperm quality due to excessive production of reactive oxygen species (ROS). Additionally, products of the oxidative reaction, especially involving lipids (e.g., malondialdehyde - MDA), may be even more harmful to sperm. Carnosine, previously reported to be present in seminal plasma of several species, may be a key factor on sperm tolerance to biotechniques by counterattacking the deleterious influence of MDA. Therefore, the aim of this study was to measure the levels of carnosine present in equine seminal plasma and relate these findings with sperm function and oxidative status during cooling and cryopreservation. Thus, semen samples were collected from 40 stallions in duplicate (N = 80) and then submitted to cooling and cryopreservation. Samples were then allocated into groups of high and low tolerance to refrigeration and cryopreservation (bad cooler and good cooler/bad freezer and good freezer, respectively), and in groups of different concentrations of carnosine (High, Medium-high, Medium-low and Low carnosine). Samples were evaluated for sperm kinetics patterns, function of sperm structures and oxidative status. In good cooler samples, it was observed higher concentrations of carnosine (Good cooler: 224.98 ±â€¯19.16 ng/mL; Bad cooler: 159.72 ±â€¯15.99 ng/mL; p = 0.0056), ROS production (Good cooler: 26.40 ±â€¯18.33%; Bad cooler: 18.33 ±â€¯1.84%; p = 0.001) and lipid peroxidation rates (Good cooler: 193.23 ±â€¯18.22 ng/mL; Bad cooler: 131.92 ±â€¯12.25; p = 0.0064). Groups of samples with higher carnosine concentrations had lower levels of malondialdehyde (High: 79.33 ±â€¯6.72 ng/mL; Medium-high: 140.45 ±â€¯11.70 ng/mL; Medium-low: 202.57 ±â€¯16.30 ng/mL and Low: 231.02 ±â€¯32.35 ng/mL; p < 0.05), demonstrating that carnosine was effective in removing lipid peroxidation products. Due to the removal of seminal plasma during the cryopreservation process, no differences occurred in carnosine levels between bad and good freezer groups. In this context, this study provides relevant data for future therapies using carnosine during cryopreservation, aiming to replace the levels lost due to the necessary removal of seminal plasma.

Effects of the utilization of homeopathic elements in commercial diluent on swine sperm viability.

It has been speculated that the homeopathic treatment of sperm cells in order to improve semen quality could be promising. However, few data is available and its use in spermatozoa requires investigation. It is well established that mitochondrial membrane potential is an important viability parameter of spermatozoa and it is intimately related to reproductive efficiency. In this manner, new technologies in order to improve the activity of sperm cells and, finally, the fecundity of swine herds are of extremely importance. Due to the lack of knowledge of homeopathic treatment effect on spermatozoa, the aim of the present study was to verify the effect of three different homeopathic treatments on viability of boar sperm cells. Three homeopathic treatments composed by Pulsatila CH6, Pulsatila and Avena CH6, Avena CH6 and one control treatment (sucrose) were added to diluted boar semen, which were cooled for 24 or 48 h. Interestingly, no positive effect of homeopathic treatments was observed over semen viability. However, it was demonstrated that the 24 h of cooling storage provided more viable sperm cells when compared to the 48-h period. This effect of storage period on sperm viability was assessed by intact plasmatic membrane, intact acrosome and mitochondrial membrane potential evaluation.