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Open Access
Comparative evaluation of different extenders of bull semen stored under different conditions
(2015-07-16) Raseona, Andrea Motswetla; Barry, D. M.; Nedambale, T. L.
Preservation of semen is an important process to ensure that semen quality is sufficient for use in assisted reproductive technologies. This study evaluated the effectiveness of three different extenders to preserve bull semen stored under different conditions, as an alternative to frozen-thawed semen straws used for artificial insemination. Semen samples were collected from two Nguni bulls using an electro-ejaculator and transported to the laboratory at 37 °C for evaluation. Pooled semen was first aliquoted into three extenders namely Triladyl, Ham’s F10 and M199 at a dilution ratio of 1:4 (semen:extender), and then stored at controlled room temperature 24 °C. Secondly pooled semen was aliquoted into four groups of Ham’s F10 extender and diluted at a ratio of 1:4, then stored at 24 °C, 17 °C, 12 °C and 5 °C respectively. Sperm motility rates were analysed after 0, 24, 48 and 72 hours. Morphology, viability and sperm DNA fragmentation were analysed after 72 hours. The study was replicated four times and data was analysed by ANOVA. Triladyl had higher sperm viability rate and total motility rate for 72 hours (P<0.01). However, Ham’s F10 had higher progressive motility rate as compared to the other extenders. There was no significant difference (P<0.01), in the viability rate between Ham’s F10 and M199. No significant difference was also observed in total sperm abnormalities (absent tails, coiled tails and bent tails), except for reacted acrosomes (P>0.05), between the two Nguni bulls. Lower temperatures than 24 °C influenced sperm motility and viability in Ham’s F10. There was no significant difference in sperm DNA fragmentation rates (P<0.01), between all the four storage temperatures which indicated that temperature did not have an influence on sperm DNA fragmentation. In conclusion, bull semen can be preserved in Triladyl or Ham’s F10 and M199 culture media stored at 24 °C and stay alive for 72 hours. Triladyl proved to be the best suitable extender showing higher sperm viability and total motility rates as compared to Ham’s F10 and M199. Lower temperatures than 24 °C noticeably decreased sperm motility and viability in Ham’s F10 culture medium.
Open Access
Comparison of progestone, PGF2A & NOVEL NC SYNCH GnRH based synchronization protocols in boer and indigenous goats of South Africa
(2016-02-10) Dara, Onayi Brighton; Barry, D. M.; Baloyi, J. J.; Dondofema, Farai
Open Access
Comparison of two different media and assisted hatching techniques on the embryo hatching rate using the mouse as a model
(2017-05-18) Negota, Nkhumeleni Cathbert; Barry, D. M.; Nedambale. T. L.
The use of in vitro culture media and assisted hatching techniques remain a challenging obstacle to hatching of blastocyst-stage embryos. Mechanical, chemical, enzymatic thinning and laser assisted techniques have been used previously, but there is still a lack of information on its application and implication in livestock. The aim of this study was to compare the effect of two in vitro culture media ((Ham’s F10 and Tissue Culture Medium 199 (TCM-199)) and four assisted hatching techniques (mechanical, chemical, enzymatic and laser) on blastocyst formation and hatching rate using murine embryos as a model. The C57BL/6 and BALB/c mouse breeds were bred and raised until they reach maturity and then bred naturally to produce a hybrid F1 generation. The light in the breeder house was controlled at 14 hours light and 10 hours darkness. Feed and water were provided ad libitum for the mice. Mature female mice were super-ovulated using equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). A total of 400 blastocysts were collected from the F1 generation and these were allocated equally for the four assisted hatching techniques (laser, mechanical, chemical and enzymatic) as well as a non-treated control group. The blastocysts were paired into a group of 10 and replicated 4-four times for each assisted hatching techniques and control group. The embryos were then cultured for 24 hours and the hatching of the embryos were observed. Hatched embryos were stained for blastomere counting. The general linear model (GLM) of statistical analysis software (SAS) version 9.4 was used to analyze the data. Assisted hatching techniques (laser, mechanical, enzymatic and chemical) yielded 46.86±37.12; 51.07±40.19; 39.05±35.83 and 33.32±37.50% of hatching, respectively under in vitro culture in Ham’s F10. There was a significant difference (p<0.05) observed between assisted hatching techniques using Ham’s F10 as culture medium. In the TCM-199, laser, mechanical, enzymatic and chemical assisted hatching techniques yielded 56.25±43.30; 52.55±35.50; 49.16±37.50 and 33.85±35.50%, respectively, with significant differences (p<0.05). However, the hatching rate of embryos for all techniques was higher when in vitro cultured in TCM-199 compared to those cultured in Ham’s F10, and statistically higher than the control group. In conclusion, laser assisted hatching technique is the best of the techniques to use to assist the hatching of murine embryos and TCM-199 is the best of the two in vitro culture media for the hatching percentage.
Open Access
Effect of bioxcell and triladyl extenders and removal of seminal plasma of equilibrated and cryopreserved goat semen
(2017-05-18) Nethenzheni, Livhuwani Pertunia; Barry, D. M.; Nedambale, T. L.
The objectives of the study were to evaluate the effect of two extenders (Triladyl® and Bioxcell®) and the removal of seminal plasma on goat buck semen. Six ejaculates were collected from six indigenous bucks by means of electro-ejaculator method, and semen was pooled, and replicated 10 times. Raw semen were 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. All six groups were analysed for spermatozoa motility rates using computer-aided sperm analysis (CASA). The spermatozoa viability for all groups were assessed using Eosin-Nigrosin, acrosome integrity using Spermac, chromatin structure using Acridine Orange, and mitochondria using JC-1 staining solutions. Both the Triladyl® and Bioxcell® washed semen groups were diluted (1:4) with Phosphate Buffered Saline (PBS) then centrifuged at 1500 x g for ten min and seminal plasma was aspirated using 1 mL sterile plastic pipette. Semen samples were diluted (1:4) as follows: Triladyl® (washed and non-washed) or Bioxcell® (washed and non-washed) and then equilibrated at 5 ºC for 2 hours. Following equilibration, semen parameters were analysed. Thereafter, the semen samples were loaded into straws and placed 5 cm above a liquid nitrogen vapour for 10 min, and then stored at -196 ºC until use. Following one month of storage, frozen semen straws per treatment group were thawed at 37 ºC for 30 seconds, then semen parameters were analysed again. 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. Total Spermatozoa motility rate of Bioxcell® (92.5±4.6), (68.2±13.5) and Triladyl® (94.9±5.5), (63.1±15.1) were significantly reduced (P < 0.05) following equilibration and freeze-thawing process, respectively on washed semen groups. Live and normal spermatozoa percentages were drastically reduced in Bioxcell® (5.2±4.9) and Triladyl® (6.9±8.6) washed semen groups, following freeze-thawing. There was a significantly lower number of spermatozoa with high mitochondrial membrane potential in non-washed semen extended with Triladyl® (68.7±26.8) compared to non-washed semen extended with Bioxcell® (49.8±20.1) following the freeze-thawing process. In conclusion, the freezing-thawing process did reduce the indigenous buck semen parameters irrespective of removal or non-removal of seminal plasma. However, Bioxcell® extender was found to be more suitable for preserving spermatozoa during equilibration and freezing/thawing process of buck semen.
Open Access
Effect of different culture media and incubation methods on culturing murine embryos in vitro using a semen straw as an alternative receptacle
(2016-05) Madzhie, Lufuno Rosheen; Barry, D. M.; Nedambale, T. L.
See the attached abstract below
Embargo
Effect of spermatozoa viability, culture receptacle, incubation, and intracytoplasmic sperm injection on vitro production of cattle embryoa using epididymal spermatozoa
(2022-07-15) Raseona, Andrea Motswetla; Barry, D. M.; Owiny, O. D.; Nedambale, T.L.
In an event of an unexpected death, injury, or inability of a bull to serve, spermatozoa can be harvested from the epididymides to enable the propagation of valuable germplasm from genetically superior males. Recovering bull spermatozoa from the epididymis of dead breeders presents the last opportunity to use the gametes and therefore, the need for the most efficient preservation and utilization of the recovered spermatozoa is paramount. One of the unique ways of preserving the male genetic material of cattle is the use of assisted reproductive technologies. The present study focused primarily on the production of in vitro cattle embryos using epididymal spermatozoa by firstly seeking the effective spermatozoa extender and preservation method, and secondly by assessing the effects of culture receptacles and incubation methods following intracytoplasmic sperm injection. In a series of experiments, ejaculated and epididymal spermatozoa were collected, chilled, cryopreserved, and used for in vitro embryo production. The viability of chilled bovine spermatozoa recovered from the epididymis stored at 5 °C for 24 h and diluted with Triladyl® or modified Ham’s F10 was assessed in the first experiment. The short-term preserved (120 h at 5 °C) ejaculated spermatozoa demonstrated a higher motility rate than epididymal sperm, however, epididymal spermatozoa harvested immediately after bull slaughter, extended with Triladyl® was found to have a higher motility rate (P < 0.05) than spermatozoa harvested 24 h post chilling of the epididymides at 5 °C diluted in modified Ham’s F10 culture media. A significant decline in viability was also demonstrated after 72 h of chilling at 5 °C with the least percentage of live cells detected with modified Ham’s F10 extended epididymal spermatozoa harvested 24 h post-mortem. Furthermore, less than 10% of acrosome defects were demonstrated across all the spermatozoa samples. It was concluded that refrigeration of epididymides at 5 °C for 24 h before the recovery of spermatozoa cells was able to sustain good spermatozoa motility when Triladyl® extender was used. The viability of frozen-thawed bull semen collected from the bull’s ejaculate and cauda epididymis was also evaluated. Similar to the observations on spermatozoa chilling in the first experiment, ejaculated spermatozoa had a higher post-thaw motility rate (P < 0.05) than epididymal sperm. However, no significant difference in post-thaw motility rate was demonstrated amongst epididymal spermatozoa recovered immediately and 24 h post-mortem (P > 0.05). The post-thaw percentage of acrosome defects, as well as the live cells, was found to be similar for both ejaculated and epididymal sperm. It was concluded that cooling of epididymides at 5 °C for 24 h before the recovery of spermatozoa cells was efficient in preserving post-thaw spermatozoa quality. In the third experiment, the success rates of in vitro embryo production using epididymal bull sperm, French semen straws as culture receptacle, and alternative incubation method was evaluated. The experiment was carried out as a completely randomised design arranged in a 3 x 2 x 2 factorial. The results demonstrated that short-term preserved (120 h at 5 °C) ejaculated spermatozoa produced a higher percentage of in vitro embryos compared to epididymal spermatozoa. However, no significant difference (P > 0.05), in blastocyst development was demonstrated between epididymal spermatozoa retrieved immediately and after 24 h post-mortem, extended in either Triladyl® or modified Ham’s F10, chilled for either 24 or 48 h. Additionally, no significant difference was demonstrated when comparing individual spermatozoa samples using French semen straws as culture receptacles, incubated in the goat doe vagina, versus a standard 5% CO2 incubator. It was concluded that in vitro embryos up to 8 days of development, cultured in French semen straws and incubated in the goat doe vagina, can be produced after fertilization with epididymal bull spermatozoa recovered immediately or after refrigeration of the epididymides at 5 °C for 24 h. Lastly, the study investigated the effect of using French semen straws as embryo culture receptacle and goat doe vagina as an alternative incubator, on in vitro embryo production following intracytoplasmic sperm injection with epididymal spermatozoa. The injected oocytes with epididymal spermatozoa recovered after bull slaughtering or after 24 hours post-mortem, cultured inside French semen straws or micro-droplets, had no difference in embryonic development rates (P > 0.05). Furthermore, when both the standard CO2 incubator and the goat doe vagina were used, no difference in embryo development stages was identified (P > 0.05), with the exception of the cleavage stage where the injected oocytes incubated in goat doe vagina showed lower rates (P < 0.05) as compared to those incubated in the standard CO2 incubator. It was concluded that fertilization and blastocyst development can be accomplished through intracytoplasmic sperm injection with cryopreserved bull epididymal spermatozoa recovered immediately or 24 h post-mortem, using French semen straws as culture receptacles and goat doe vagina as an alternative incubator.
Open Access
The efficiency of ultrasonorgraphy in monitoring ovarian structures and foetal development in goats, sheep and cattle as verified through laparoscopy and laparotomy
(2018-05-18) Siphugu, Steven Mbonalo; Barry, D. M.; Mashiloane, M. L.
The main purpose of this study was to assess the efficiency of ultrasonography in monitoring reproductive organs, pregnancy diagnosis, and foetal gender identification and to verify its reliability by laparoscopy and laparotomy, where applicable. Reproductive organs, pregnancy diagnosis and gender of the foetus were examined by A-mode ultrasound using 3.0 - 8.0 MHz trans-rectal transducer. A Sony Olympus Model laparoscope with a camera transducer was used to monitor the reproductive organs and pregnancy diagnosis. In monitoring the follicular dynamics, daily ultrasonography (ULTS) scanning was done for 17 days in sheep and for 21 days in both goats and cattle. Follicles of diameter ≥ 3 mm were selected for analysis of growth, ovulation and regression. For determining the efficiency of the techniques, laparoscopy (LAPSC) and laparotomy (LAPT) were used on days 3 and 10 of the goats and sheep oestrous cycle. The follicles were grouped into three categories according to their diameter as 3 - 4.9 mm, 5 - 7.9 mm and ≥ 8 mm, whereas the follicles of cattle were grouped as 3 - 4.9 mm, 5 - 9.9 mm and ≥ 10 mm. Early pregnancy diagnosis examinations were carried out from day 18 post insemination until pregnancy was confirmed. Foetal gender examinations were conducted from day 40 of pregnancy until the day the gender of the foetus was confirmed. Follicular development was accompanied by the occurrence of waves of follicular growth at different period of the oestrous cycle. The first follicular wave emerged on day 1.0 ± 0.4 in goats, 1.2 ± 0.4 in sheep and 2.2 ± 0.4 in cattle. The maximum diameter of the dominant follicles of observed follicular waves in goats was 7.3 ± 0.4 mm, 6.6 ± 0.2 mm, 7.3 ± 0.2 mm; in sheep was 6.4 ± 0.4 mm, 6.6 ± 0.4 mm and 6.7 ± 0.7 mm and in cattle was 13.1 ± 0.8 mm, 14.2 ± 0.6 mm and 15.7 ± 0.6 mm in wave 1, 2 and 3, respectively. However, the maximum size of the dominant follicle of the ovulatory wave in cattle was larger than the dominant follicles of both first and second waves, but in goats and sheep the dominant follicles were of similar size throughout the waves. In cattle, the ovulatory wave was shorter (p ˂ 0.05) than the duration of the first and second waves, while in sheep and goats were similar throughout the waves. In goats the total number of follicles counted in right and left ovaries under category 3 - 4.9 mm was lower with ULTS and LAPSC than with LAPT method (p ˂ 0.05). In sheep the mean number of follicles between 3 - 4.9 mm category in both right and left ovaries were different (p ˂ 0.05) between ULTS and LAPT. However, for categories 5 - 7.9 mm and ≥ 8 mm in both goats and sheep the mean numbers of follicles observed by all techniques were similar (p ˃ 0.05). In goats, pregnancy diagnosis accuracy improved from zero percent on day 18 to 100% on day 26 - 28, in sheep pregnancy diagnosis was 40% on day 18 and improved to 100% on day 20 - 22 vi of gestation. In cattle accuracy of pregnancy diagnosis was not possible at day 18 and gradually increased to 100% on day 30 - 32 of gestation. Out of 5 (100%) goat’s foetuses whose gender was determined, the diagnosis was correct in 100% (3/3) of the male foetuses and 100% (2/2) of the female foetuses. In sheep two foetuses were sexed as males while the other three were sexed as females and were both 100%. Out of 60% (3/5) of foetuses examined in cattle, 1 (100%) was identified as male and the remaining 2 (100%) were identified as females. The results obtained confirmed that the accuracy for foetal gender by ultrasonography was 100% in all foetuses observed. The current study demonstrated that trans-rectal ultrasonography examination is an efficient method for monitoring follicular dynamics, diagnosing pregnancy and foetal gender identification and that it is as reliable as laparoscopy and laparotomy where they were applied together.
Open Access
Evaluation of Nguni bull semen-extended in tris egg yolk extender, soybean milk and coconut water based extenders and stored at different temperatures
(2017-09-18) Mayombo, Pie Veillard Kalonji; Barry, D. M.; Owiny, D. O.
In order to realize many of the potential advantages of AI, storage of semen is necessary. Semen storage is only possible using a system that decreases and/or halts the metabolic processes of the spermatozoa, allowing no significant loss of fertility. Numerous factors affect the success of spermatozoa storage. This study was designed to compare the effects of egg yolk, soybean milk and coconut water in Tris extender using different storage methods for Nguni bull spermatozoa storage. Bull semen was collected from two adult Nguni bulls approximately four years old and kept under similar managerial conditions. Using electro-ejaculator, semen was collected from each bull into a graduated semen collection tube. Macroscopically evaluation of the sample was performed immediately after collection. Only the semen free from contamination was processed. The kinetic properties namely: total spermatozoa motility, and progressive spermatozoa motility were analysed using CASA. Semen sample was stained and spermatozoa morphology and vitality also analysed using CASA. The extended semen was then split into three groups. The first group was stored at room temperature (25 °C). The second group was cooled to 4 °C and stored in the refrigerator. The third group was also cooled to 4 °C for 2 h in the refrigerator, then held in LN2 vapour 5 cm above the surface of LN2 at ~ -80 °C for 10 min and then plunged into LN2 for storage at -196 °C. Different colours of straws and plugging powder were used for identifying each extender. After 3 days of storage at room temperature, in the refrigerator and in LN2, the extended semen was split into three portions and assayed for kinetic properties using the first portion. The second portion was assayed for spermatozoa morphology and the third portion for spermatozoa vitality. The results from the fresh semen extended with all three extenders (TEYE, SBME and COWE), and analysed immediately after dilution at room temperature (25 ºC), showed no significant difference (P > 0.05) in the mean values of the kinetic and morphologic properties and viability, on spermatozoa TM, PM, AR, AT, CT; BT and LS. After three days of storage, there was no significant difference (P > 0.05) in the kinetic morphologic properties and viability of semen stored at room and refrigeration temperature regardless of the extender in use. There were, however, significant differences (P < 0.05) in the TM, PM, AR and DL of the frozen semen samples. For the short storage period of semen used for AI, from this study, it is recommended that semen should be kept at room or refrigeration temperature regardless of the three extenders used. However, for long storage of frozen semen TEYE is recommended. The egg yolk-based extender provided greater preservation of motility and bull spermatozoa integrity during the freezing process than did SBME and COWE.
Open Access
Evaluation of suitable chilled, extended semen preservation time and their effects of different artificial insemination techniques on the fertility of indigenous Venda goats
(2017-09-18) Monyeleote, Vukosi; Barry, D. M.; Fushai, F.; Bhehbe, E.
The aims of the study were to evaluate the effects of dilution and chilled storage time on the quality of semen, and of different artificial insemination techniques on fertility in artificially inseminated indigenous Venda does. Fresh semen was collected using an artificial vagina from three Boer bucks aged 4±1.55 years once every four days during July and August 2016. Semen was pooled and samples were divided into two equal parts, which were extended using Biladyl® extender at ratios of 1:5 and 1:10 v/v (semen to extender), before refrigeration for 120 hours at 5 °C. The fresh undiluted semen and freshly extended semen were evaluated in six replicates for sperm motility, live-dead and sperm morphology using the Sperm Class Analyzer (SCA). Extended semen continued to be evaluated at 24 hour intervals for 120 hours. Ninety indigenous Venda does were obtained from different flocks in the Vhembe district and kept intensively in one 10 m x 40 m pen at the University of Venda experimental farm in the goat feedlot. The does were fed and watered ad libitum. After acclimatization for 14 days, estrus was synchronized using a controlled internal drug release (CIDR) containing 0.3 g of progesterone. Upon removal of the CIDR, does were injected 10 mg of PGF2α (Lutalyse® dinoprost tromethamine) Sterile Solution. At 24 hours after the removal of the CIDR, the does were injected intramuscularly with 300 international units (IU) of equine chorionic gonadotrophin (eCG). Forty eight hours after the removal of the progesterone, freshly collected and diluted (1:5 ratio ~150x106 sperm/ml), five day-stored semen were used to inseminate the does using cervical (CAI), trans-cervical (TAI), and laparoscopic artificial (LAI) insemination methods in a complete randomized design (CRD) with a 2 X 3 factorial arrangement of the treatments with 15 replications per treatment. The does were tested for pregnancy after 30 days using ultrasonography. Analyses of variance was performed on the pregnancy, kidding rates and on prolificacy using the GLM procedure of Minitab (Minitab 2013). Significant differences in all motility parameters were observed between the extension ratios and storage time (P<0.01). There were significant interactions between the extension ratio and storage time (P<0.05) on the sub-population of sperm cells with non-progressive motility (NON-P). Significant (P<0.01) interaction was observed between the semen extension ratio and storage time on medium and slow spermatozoa (P<0.01). The method of insemination did not (P>0.05) affect fertility, though both pregnancy and kidding rates numerically decreased in the order laparoscopic insemination (LAI)≥ trans-cervical insemination (TAI)≥ cervical insemination (CAI). Overall, 71% kidding rate was achieved.
Open Access
Evaluation of the effectiveness of different extenders of goat buck semen under refrigerated conditions.
(2015-10-14) Ajao, Olumide Adewale; Barry, D. M.; Benyi, K.
Open Access
Presevation of boer goat semen in liquid nitrogen vapour in comparison to the conventional freezing method using different extenders, freezing and thawing regimes
(2018-05-18) Kalobo, Kidinda; Barry, D. M.; Fushai, F.
The Boer goat (Capra hircus) is one of the most desirable goat breeds for meat production. The impact of cryopreservation on the viability of its semen depends on the extenders, freezing and thawing methods. This study evaluated the effects on sperm viability in Boer goat semen extended using Bioxcell, Biladyl and Ham’s F10, and frozen in semen straws placed on a rack at 4, 5, 6 or 7 cm above the surface of liquid nitrogen. After storage in liquid nitrogen for 7 days, the frozen semen was thawed at 37 oC for 30 seconds or 90 oC for 5 seconds. Samples of sperm were also frozen to -196 oC in a programmable freezer, as the control regime for the freezing treatments. Sperm morphology, motility and viability were evaluated using the computer aided sperm analysis (CASA) system in a randomised design in which the treatments were in a 3 (extender) X 5 (freezing regime) and X 2 (thawing regime) factorial arrangement. The extenders Bioxcell and Biladyl were affected in the total motility, progressive motility and static (P<0.01), the motility was overall maintained only in straws placed at 5 cm above the liquid nitrogen level, with significant difference for the interaction extender X freezing regime in the total motility (p<0.01), non-progressive motility (p<0.05) and progressive motility (p<0.01), the 37 oC for 30 sec thawing regime had significantly more (P<0.05) in cut-head spermatozoa. Ham’s F10 extender had significantly lower normal spermatozoa (P<0.05) compare to Biladyl and Bioxcell extenders. In conclusion, the extender type, freezing and thawing regime were important factors for consideration in goat semen

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