D. M. Barry’s scientific contributions

The objectives of the study were to evaluate the effect of two extenders (Triladyl® and Bioxcell®) and the removal of seminal plasma on indigenous buck’s semen. Semen was collected from six indigenous bucks using an electro-ejaculator. Raw semen was pooled and randomly allocated into six groups as follows: (i) Raw non-washed, (ii) Raw washed, (iii) Triladyl®-washed, (iv) Triladyl®-non-washed, (v) Bioxcell®-washed and (vi) Bioxcell®-non-washed. Both the Triladyl® and Bioxcell® washed semen samples groups were diluted (1:4 v/v) with Phosphate Buffered Saline (PBS) then centrifuged at 1500x g for ten min and seminal plasma was removed. The groups were analysed for spermatozoa motility rates using Computer-Aided Sperm Analysis (CASA). The spermatozoa viability was assessed using Eosin-Nigrosin, acrosome integrity using Spermac, chromatin structure using Acridine Orange, and mitochondria using JC-1 staining solutions. Semen samples were diluted (1:4 v/v) as follows: Triladyl® (washed and non-washed) or Bioxcell® (washed and non-washed) and then equilibrated at 5°C for 2 hours. Equilibrated semen samples in 0.25 mL French straws were placed 5 cm above a Liquid Nitrogen (LN2) vapour for 10 min, and stored for one month. Frozen semen straws per treatment group were thawed at 37°C for 30 seconds. Significant differences among the mean values of semen parameters were determined by Tukey’s test using ANOVA, GLM procedure of SAS version 12.1 of 2010. The spermatozoa progressive motility rate in non-washed semen extended with Bioxcell® was significantly higher (89.6±7.5a) compared with that of non-washed Triladyl®, washed Bioxcell® and Triladyl® (P<0.05). Live spermatozoa percentage in washed semen extended with Triladyl® extender was reduced (27.7±17.1) significantly compared with the other groups (P<0.05). There was a lower percentage of spermatozoa with high mitochondrial membrane potential in non-washed and washed semen extended with Bioxcell® (39.5±23.2 and 37.9±28.6, respectively) compared with that of non-washed and washed semen extended with Triladyl® (P>0.05). The spermatozoa progressive motility rate in non-washed semen extended with Bioxcell® (58.5±10.0) extender was significantly higher compared with that of the other groups (P<0.05). There was a higher live and normal spermatozoa percentage in non-washed semen extended with Bioxcell® (45.7±21.2) compared with that of the other groups (P<0.05). In conclusion, Washing of seminal plasma in semen extended with Triladyl® was not essential, as it lowered viability, progressive motility and chromatin membrane integrity prior and post-cryopreservation. However, Bioxcell® extender was found to be more suitable for preserving spermatozoa during equilibration and freezing/thawing process of non-washed and washed buck semen.

The study investigated the influence of Assisted Hatching (AH) techniques (mechanical, chemical, enzymatic thinning and laser) and two in vitro culture media (Ham’s F10 and TCM-199) on hatching rate of mice embryos (blastocyststage) following 24 hours of culture. The C57BL/6-black (n=30) and BALB/cwhite (n=30) mouse breeds were raised until maturity and naturally bred to produce a F1 generation. The light in the breeding house was controlled and the mice were fed ad libitum. Female mice (n=30) were injected (peritoneal) with 0.1 ml (5 IU) of eCG into the abdominal cavity with 1 ml syringe and 0.5x16 mm needle to stimulate follicular growth and 46-49 h later was injected with 0.1 ml (5 IU) of hCG to cause ovulation, maintain the corpus luteum and stimulate it to secrete progesterone for maintenance of pregnancy. After the injection, the male and female (1:1) could mate overnight. Female mice with vaginal plugs were observed and kept separately for blastocyst-stage embryos collection on day three following successful mating. They were euthanized, and abdomen sterilized with 70% ethanol. Using a sterile surgical scissor, a fine cut was made, holding the skin firmly above and below the incision, the skin was pulled apart using forceps. The embryos were flushed from the uterus using a 30-gauge needle with culture media. Following the AH techniques, embryos were cultured in TCM-199 or Ham’s F10 for 24 hours and zonal thickness of all hatched embryos were measured. Immediately after assisted hatching, the embryos were cultured into two different in vitro culture media. All embryos hatched were stained and the zonal thickness of embryos were measured. The number of blastomeres were counted and recorded 24 h later. Data collected were subjected to analysis of variance using PROC General Linear Model. The Tukey’s test was used to separate the means. A significant difference was observed between the thickness of Zona Pellucida (ZP) pre and post treatment after 24 hours of culture. However, there was no significance difference among blastocyst hatching rate and the blastomeres nuclei counted after staining. The thickness of the ZP decreased with individual AH techniques. The interaction between AH techniques and in vitro culture was found to be significantly different on blastocyst hatchability. However, laser AH technique had highest hatchability (56.3%) when embryos were cultured in TCM-199 followed by mechanical AH techniques (52.6%). The hatchability rate (33.3%) was recorded in the chemical AH technique group. The blastomeres nuclei counted under interaction of AH techniques and culture media was not differently affected, with the values ranging from 69 to 76%. In conclusion, the use of different AH techniques resulted in varying effect and increase outcomes towards the hatching rate.

The present study was undertaken to assess viability of frozen-thawed bull semen collected from the bull's ejaculate and cauda epididymis. A total of 30 ejaculates were collected from three bulls twice per week for 5 weeks (Control). Caudal epididymis were collected from slaughtered beef cattle of unknown origin from the local abattoir. Caudal epididymal sperm was recovered immediately after slaughtering (EP-0 h) and after cooling at 5°C for 24 h (EP-24). The epididymal and ejaculated samples were each pooled together before being extended with Triladyl. Diluted samples per treatment were loaded into a 0.25-mL French straw and cooled to 5°C in 4 h. Cooled straws were placed 4 cm above liquid nitrogen to freeze for 10 min. Frozen straws were immersed into LN2 and kept for 7 days at -196°C. Samples were analysed immediately after dilution and post-thawing using the computer aided sperm analysis for sperm motility rate, viability and acrosome defects. The highest sperm motility rates were observed with EJ-0 h before and after cryopreservation. However, the difference in sperm motility parameters between EP-0 h and EP-24 h evaluated before and after freezing was not significant (P > 0.05). Furthermore, no significant difference in live cells mean values was observed between the three samples on freezing (P > 0.05). In relation to spermatozoa acrosome defects, there was no significant difference observed among the three samples before and on freezing (P > 0.05). In conclusion, the results from the present study revealed that cooling of epididymides at 5°C for 24 h before the recovery of sperm cells was efficient in preserving epididymal sperm viability. However, ejaculated bull spermatozoa had higher sperm motility and viability rate than epididymal sperm.

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.

The in vitro culture media and assisted hatching techniques remain challenging obstacles to be utilised widely. Mechanical, chemical, enzymatic thinning, and laser-assisted techniques have been used previously but information is still lacking on its application in livestock. The aim of this study was to compare the effect of 2 in vitro culture media (Hamster F10 and TMC-199) and 4 (mechanical, chemical, enzymatic, and laser) assisted hatching techniques on blastocyst formation and hatching rate using murine embryos as a model. The C57/b and Balb/c breeds were raised until they reached maturity and bred naturally to produce F1 generation. The light in the breeding house was controlled at 14 h light and 10 h dark. Feed and water were provided ad libitum for the mice. Superovulation of females were stimulated using equine chorionic gonadotropin and human chorionic gonadotropin. The F1 generation was used for the collection of the 400 blastocysts and randomly allocated into 4 assisted hatching techniques. Blastocysts were paired into a group of 10 and replicated 4 times for each assisted hatching technique. The general linear model of SAS version 9.4 (SAS Institute Inc., Cary, NC, USA) was used to analyse the data. Assisted hatching techniques of laser, mechanical, enzymatic, and chemical yielded 46.9 ± 37.1, 51.1 ± 40.2, 39.1 ± 35.8, and 33.3 ± 4.5%, respectively, under in vitro culture of Hamster F10. The TCM-199, laser, mechanical, enzymatic, and chemical assisted hatching techniques yielded 56.3 ± 43.3, 52.6 ± 35.5, 49.2 ± 37.5, and 33.9 ± 35.5%, respectively, with a significant difference. There was no significant difference observed in assisted hatching techniques and Hamster F10 culture medium. However, the hatching rate of embryos for all techniques was higher when in vitro cultured in TCM than cultured in Hamster F10. Hatching rate of blastocysts increased from chemical, enzymatic, mechanical, and laser with response to Hamster F10 and TCM; thus, laser is a suitable assisted hatching technique with TCM-199.

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.

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.

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.