الفهرس 
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Abstract
The present study was designed to investigate:
1. Follow-up of bull semen processing and CR based on using the processed post-thawed bovine semen under different environmental conditions
For this purpose, all semen processing steps were followed in the AI center, Beni-Suef Governorate, Egypt from period (January to June 2021), ejaculate volume, density, dilution rate, dilution volume, mass activity, individual motility, individual motility after cooling and individual motility after freezing were evaluated according to each bull and then data resulting from insemination with the processed semen was gathered from villages in the Beni-Suef Governorate. Calculations were made for the number of services per conception and CR.
The statistical analysis for semen processing and fertility results was done using independent samples T-test (* Significant difference at P ˂ 0.05, ** significant difference at P ˂ 0.01, and *** significant difference at P ˂ 0.001).
The results of the present study revealed a significant difference in the percentage of post-thawing individual motility between winter and spring, with a higher value (51.14±0.43) recorded during the winter while there is no significant differences were observed in the other semen processing parameters by timing. According to the CR results, the findings revealed that there is a little variation between the winter and spring months concerning the rate of conception (The statistical analyses revealed a higher CR in winter compared to spring); rather, the significance was seen most notably in terms of the number of cows conceived with second and third inseminations, with larger values (0.16±0.04; 0.1±0.38) being recorded during the winter season respectively.
2. Effect of CSNPs and aloe vera on capacitation and acrosome reaction of bovine spermatozoa during in vitro and 3. Effect of capacitated bovine spermatozoa with CSNPs and aloe vera on fertilization rate of bovine oocytes.
In order to achieve that, the following steps were done:
The z-average particle size, zeta potential analysis, and yield of CSNPs were determined.
With regard to CSNPs, Three concentrations of CSNPs (10, 20, and 100 µg/ml) were prepared. Motile spermatozoa were separated from frozen-thawed semen by a swim-up technique and capacitated in Sperm-TALP medium with different concentrations of CSNPs, without treatments (Positive control) “Sperm cells + SP-TALP containing heparin and devoid of CSNPs” and without heparin (Negative control) “contains sperm cells and nanoparticles only without heparin”. Sperm cells were incubated for 90 minutes at 39°C in a 5% CO2 incubator. Sperm viability, HAM of spermatozoa and AR were evaluated every 30 minutes interval.
For aloe vera gel, two concentrations (5 and 10µg/ml) were prepared. Motile spermatozoa were separated from frozen-thawed semen by a swim-up technique and capacitated in Sperm-TALP medium with different concentrations of aloe vera, without treatments (Positive control) “Sperm cells + SP-TALP containing heparin and devoid of aloe vera” and without heparin (Negative control) “contains sperm cells and aloe Vera only without heparin”. Sperm cells were incubated for 90 minutes at 39°C in a 5% CO2 incubator. Sperm viability, HAM of spermatozoa and AR were evaluated every 30 minutes interval.
Cattle ovaries were collected from the slaughterhouse and transported to the laboratory within 2 hours after slaughtering.
In the laboratory, follicles aspirated from the ovaries, number, and state of COCs were recorded.
Oocytes were cultured in each droplet (100µl) of the maturation medium in a sterile petri dish which was covered with mineral oil and incubated for 22-24 hours in a CO2 incubator (5% CO2) at 390C.
The rate of oocyte maturation was evaluated after 22-24 hours of incubation by cumulus expansion.
The matured oocytes were washed three times in IVF-TALP and then transferred to droplets of IVF-TALP medium (10-12 oocytes/100 µl), inseminated with prepared treated frozen-thawed bull sperm and incubated at 390C in 5% CO2 incubator for 24h and then examined for evidence of fertilization.
The obtained data of sperm capacitation was statistically analyzed by using two-way ANOVA followed by LSD for multiple comparisons while the data for in vitro fertilization parameters were conducted by one-way ANOVA.
Analysis of data revealed that:
(A) According to the effect of CSNPs on the capacitation of bovine spermatozoa and consequently the fertilization of bovine oocytes:
The overall proportion of spermatozoa with PM had decreased across all groups over time, with a significantly lower value (3.69±2.57); mean ± SD was observed at the CSNPs concentration of 20µg/ml as compared to other treatments.
A significantly higher value (29.81± 0.91/34.69± 1.13) for HAM and AR respectively, was recorded at a CSNPs concentration of (20µg/ml) after incubation time (60 minutes) when compared with other treatments.
The long incubation period showed a negative influence on sperm HA and AR.
A significant effect of CSNPs on the percentage of live zygotes with the highest value (7.69± 2.96) was observed at a CSNPs concentration of (20µg/ml) when compared with other treatments.
Concerning the effect of CSNPs on the development rate of cultured zygotes with male and female pronuclei, second polar body as well as 2 cell stages, the results showed a significantly higher value (0.50±1.09, 2.25 ± 2.23 and 0.13±0.34) respectively at CSNPs concentration of (20µg/ml) than other treatments.
The results also show a significant effect of CSNPs on the percentage of non-developed (degenerated) zygotes with the lowest value (4.56±1.59) observed at CSNPs concentration of (20µg/ml) when compared with other treatments.
(b) According to the effect of aloe vera on the capacitation of bovine spermatozoa and consequently the fertilization of bovine oocytes:
The overall percentage of spermatozoa with PM had declined across all groups by time progressing with a significantly lower value (1.06±0.92); mean ±SD was found at the aloe vera concentration of (10µg/ml) when compared with other treatments.
A significantly higher value (28.31±2.30 /33.81±2.16) for HAM and AR respectively, was recorded at an aloe vera concentration of (10µg/ml) after incubation time (60 minutes) when compared with other treatments.
The long incubation period showed a negative influence on sperm HA and AR.
Aloe vera concentration of (10µg/ml) had a significant impact on the number of viable zygotes, with the highest value obtained (8.50±2.36) when compared to other treatments.
Concerning the effect of aloe vera on the development rate, it was found that the addition of aloe vera (10µg/ml) resulted in a significantly higher value for cultured zygotes with male and female pronuclei, second polar body, and oocytes cleaved to two cell stages (0.50 ± 0.81; 3.94 ± 2.67; 0.31 ± 0.60) respectively than other groups.
Furthermore, The data also revealed that aloe vera has a substantial influence on the number of non-developed (degenerated) zygotes, with the lowest value (3±1.26) seen at aloe vera concentration of (10µg/ml) when compared to other treatments.
Our study has shown that using chitosan nanoparticles at a concentration of 20µg/ml and aloe vera at a concentration of 10µg/ml, with a 60-minute incubation time, can enhance the quality of sperm samples in assisted reproduction techniques such as in vitro fertilization. These treatments act as antioxidants and show promise in resolving issues related to poor sperm quality during IVF.