O. A. Ajao’s scientific contributions

The aim of this study was to evaluate the viability of bull spermatozoa diluted with commercial semen extender and two culture media stored at controlled room temperature (24 degrees C) for 72 hours. Two Nguni bulls were used for semen collection with the aid of an electro-ejaculator. After macroscopic evaluation, semen was pooled and aliquoted randomly into Triladyl, modified Ham's F10, and TCM-199 culture media, and then stored at 24 degrees C. Sperm motility parameters, morphology, and viability were analysed with computer aided sperm analysis (CASA) after 0, 24, 48 and 72 hours. The study was replicated four times, and data were analysed using analysis of variance (ANOVA). Triladyl had significantly higher sperm viability rate (41.3%) and total motility rate (96.3%) for 72 hours than modified Ham's F10 (86.8%; 26.5%) and TCM-199 (76.7%; 25.0%) culture media. Ham's F10 had higher progressive motility rate (37.8%) than the other extenders TCM-199 (31.7%) and Triladyl (23.4%). There was no significant difference in viability rate between Ham's F10 (26.5%) and TCM-199 (25.0 %) after 72 hours' storage at 24 degrees C. Furthermore, no significant difference was observed in total sperm abnormalities, except for reacted acrosomes and absent tails, between the two Nguni bulls. Nguni semen can be preserved in Triladyl or modified Ham's F10 and TCM-199 culture media, stored at 24 degrees C, and stay viable for 72 hours.

In vitro fertilization in the straw system might increase the efficiency of fertilization and the quality of blastocyst formation as compared with micro-drops-IVF systems. The aim of the study was to in vitro fertilize mouse oocytes and culture the resulting zygotes in bi-gas incubator and in a goat vagina and compare the in vitro embryo developmental stages in TCM-199 and Ham’s F10 culture media until the blastocyst-stage of development. F1 generations (Balb C × C57) were used to harvest oocytes and spermatozoa. The fresh sperm were capacitated in different incubation methods (bi-gas incubator and in the vagina of a goat). A volume of 2–4 µL of Ham’s F10 containing capacitated sperm (~8 × 106 per mL) were placed into Ham’s F10 fertilization drops under the oil, containing 10 oocytes and penicillamine, hypotaurine, and epinephrine for enhancing sperm motility and penetration of oocytes. The same procedure was used with the TCM-199 medium and IVF drops without oil (both TCM-199 and Ham’s F10) for straw filling. The presumptive embryos in Ham’s F10 and TCM-199 were divided into different groups: first group were cultured in micro-drops, second group the embryos were aspirated in semen straws and placed in the incubator (incubator straws) for culture, and other straws were covered with a sponge and inserted in the vagina of a goat (vaginal straws) for culture. The resulted blastocysts were stained using Hoechst 33528 solution and blastomeres were counted on a fluorescent UV light inverted microscope at 400× magnification (Nikon Eclipse TI, Narishige Co., Ltd., Amityville, NY, USA). The results were analysed by 2 × 2 factorial designs and Student’s t-test was used to separate the mean. There was no statistical difference (P > 0.05) between the media and incubators on the stage of murine embryo development. The overall fertilization rate was 94 to 99%. The incubator straws with Ham’s F10 (80.5%) had the highest rate of embryos that reached the blastocyst stage, followed by incubator straws with TCM-199 (77.0%), and vaginal straws with Ham’s F10 (60.0%) had the lowest rate of embryos that reached the blastocyst stage. The overall mean number of blastomeres in the blastocyst stage of the embryos ranged from 85 ± 9 to 90 ± 9 cells in all receptacles and incubators. It was concluded that the fertilization and culturing of murine embryos are possible in straws incubated in a bi-gas incubator and in the goat vagina as an alternative method of fertilizing oocytes and culturing murine embryos. In addition, Ham’s F10 and TCM-199 can both be used to fertilize oocytes and culture murine embryos until blastocyst formation embryo in vitro, incubated in a bi-gas incubator or in the vagina.

This study investigated the correlations between methods of assessing sperm qualities; namely, sperm total motility (TM), sperm vitality, acrosome integrity, DNA integrity, and sperm membrane integrity. A total of 60 ejaculates from 6 bucks were collected and the spermatozoa evaluated. The overall mean percentages of sperm TM, sperm vitality (eosin-nigrosin and propidium iodide stains), sperm acrosome integrity (Spermac and SpermBlue® stains), sperm DNA integrity (acridine orange and halotech), and sperm membrane integrity (hypoosmotic swelling test and water test) were 94.7 ± 0.5, 81.0 ± 0.6. 79.6 ± 3.7, 79.7 ± 1.8, 78.6 ± 5.3, 75.7 ± 5.5, 74.3 ± 5.1, 73.1 ± 3.5, and 73.4 ± 3.6, respectively. There were significant correlations (P < 0.05) between mean percentage live sperm evaluated with eosin-nigrosin stain and sperm TM (r = 0.813), between percentage intact acrosome assessed with SpermBlue® and sperm TM (r = 0.846), and between SpermBlue® and eosin-nigrosin (r = 0.965). There were highly significant correlations (P < 0.01) between sperm membrane integrity evaluated with HOS test and sperm TM (r = 0.871), between percentage of intact sperm DNA assessed with halotech and SpermBlue® (r = 0.832), and between percentage of intact spermatozoa DNA assessed with acridine orange and percentage intact acrosome evaluated with spermac stain (r = 0.862). Under the conditions of this study, the correlated methods of sperm analysis proved suitable for analysis of goat spermatozoa and can serve as useful indicator of potential fertility for sperm. They could be used for accurate assessment of the individual sperm cell rather the population as a whole. Motility, eosin-nigrosin stain, SpermBlue®, halotech and acridine orange stain still remain practical and valuable tools for predicting sperm fertilizing ability.

The removal of seminal plasma has a significant effect on semen characteristics. The aim of the study was to evaluate the effect of seminal plasma removal on sperm characteristics following semen dilution with Triladyl® or Bioxcell® extenders during cryopreservation. Semen samples were collected from 6 matured South African indigenous bucks for a period of 8 weeks by means of electro-ejaculation. Semen samples were pooled and then divided into 4 aliquots (Triladyl® -washed and non-washed or Bioxcell® -washed and non-washed) and diluted (1:4 vol/vol). Assessment of sperm motility characteristics was done by computer-aided sperm analysis (CASA) technology. Evaluation of mitochondria membrane integrity was done using JC-1 staining solution. Triladyl® and Bioxcell® washed semen sample groups were centrifuged at 1500 × g for 10 min, and seminal plasma was separated from sperm pellet using 1-mL plastic pipettes. After dilution of all semen sample groups, they were cooled by placing tubes into water (25°C) and then immediately placed in a 5°C fridge for equilibration for 2 h. At the end of equilibration period, all aliquot semen sample (final dilution concentration of 150 × 10⁶ sperm/mL) groups were loaded into straws (0.25 mL) per treatment groups and placed horizontally 5 cm above the liquid nitrogen vapour for 10 min. At the end of freezing process, all semen straws per group were plunged directly into liquid nitrogen (-196°C) and stored until thawed. Frozen-thawed semen samples per treatment were analysed for sperm motility characteristics by CASA. JC-1 staining solution was also used during evaluation of mitochondria membrane integrity of frozen-thawed semen samples per treatment. Significant differences (P < 0.05) among mean values of semen parameters were determined by Tukey’s method. Sperm total motility rate of non-washed semen in Bioxcell® (85.0 ± 3.4) and Triladyl® (73.9 ± 13.8) was significantly reduced (P < 0.05) by cryopreservation compared with fresh (98.9 ± 1.2) semen sample. There was greater (P < 0.05) sperm mitochondrial membrane damage due to cryopreservation in non-washed semen group extended with Bioxcell® (50.2 ± 20.1) compared with semen extended with Triladyl® (31.3 ± 26.8) and fresh (16.6 ± 14.2) semen sample. Sperm total motility rate in Bioxcell® (68.2 ± 13.5) and Triladyl® (63.1 ± 15.1) groups on the non-washed semen were significantly reduced (P < 0.05) following cryopreservation, compared with fresh (98.3 ± 2.7) semen sample. The percentage of sperm with damaged mitochondria membrane in washed semen was significantly reduced (P < 0.05) in Triladyl® (21.6 ± 16.8) group compared with Bioxcell® (34.7 ± 14.9) group and fresh (35.0 ± 20.8) semen sample. In conclusion, seminal plasma removal reduced sperm motility rate in both extenders (Bioxcell® and Triladyl®) following post-thaw. In addition, sperm mitochondria membrane damage was higher in non-washed semen samples extended with Bioxcell® extender.

Preservation of semen is an important process to ensure that semen quality is sufficient for assisted reproductive technologies. The aim of this study was to evaluate the viability of bull semen collected by electro-ejaculation using commercial semen extender and 2 modified culture media stored at controlled RT (24°C) for 72 h. Two Nguni bulls were used for semen collection; after collection, the semen was evaluated macroscopically for volume, pH, and colour, and microscopically for sperm motility, viability, and morphology. Uncontaminated semen samples with progressive motility >70% and morphological defects <20% were pooled after collection before being aliquoted into 3 extenders, namely Triladyl, modified Ham’s F10, and TCM-199 culture media, at a dilution ratio of 1:4 and then stored at controlled RT (24°C). Sperm motility rate was analysed using the computer-aided sperm analyser after 0, 24, 48, and 72 h of storage. Sperm morphology and viability was performed after staining the sperm cells with spermac and nigrosine-eosin stain, respectively. The study was replicated 4 times and data were analysed using ANOVA. Triladyl had a higher sperm viability rate (41.3%) and total motility rate (96.3%) for 72 h (P < 0.01) compared with the 2 modified culture media, Ham’s F10 (26.5 and 86.8%) and TCM-199 (25.0 and 86.7%), respectively. However, Ham’s F10 had higher progressive motility rate (37.8%) as compared with the other extenders, TCM-199 (31.7%) and Triladyl (23.4). There was no significant difference (P > 0.05), in viability rate between Ham’s F10 (26.5%) and TCM-199 (25.0%). No significant difference (P > 0.05) in straight line velocity was observed for the three extenders. Furthermore, no significant difference was observed in total sperm abnormalities, except for reacted acrosomes and absent tails (P > 0.05), between the 2 Nguni bulls. Nguni semen can be preserved in Triladyl or modified Ham’s F10 and TCM-199 culture media stored at 24°C and stay viable for 72 h. Triladyl proved to be the best suitable extender of the 3 extenders, showing higher sperm viability and total motility rate as compared with Ham’s F10 and TCM-199 modified culture media.