Nguni Bulls Research Articles

Superovulation Response and Embryo Quality Recovered Following Flushing Nguni Heifers and Cows

The aims of the study were to compare super ovulatory response, fertilization rate and evaluate embryo quality recovered from super ovulated Nguni stud cows and heifers. Nguni cows (n = 15) and heifers (n = 10) aged 4-6 and 2 years, respectively, were used as embryo donors. Embryo donors were synchronized for estrus before superovulation. For superovulation donors were administered with 350 mg of FSH (Folltropin®-V) divided into 2 injections daily 12 h apart for 4.5 days on a decreasing dosage. Semen was collected from two Nguni bulls (bull 1 and 2) using an Electro Ejaculator (EE) and semen characteristics (volume, motility, viability, concentration and morphology) were recorded and motility parameters were assessed by computer aided sperm analysis before Artificial Insemination (AI). Donor cows and heifers were inseminated randomly with semen straws from either bull 1 or bull 2. Embryos were flushed 7 days after AI using a nonsurgical technique and were evaluated under microscope for quality. Data were analyzed using one-way ANOVA and linear or quadratic relationships were used to determine effects of semen traits on fertilization rate and embryo quality. No significant differences were observed on the super ovulatory response rate of Nguni cows and heifers (P>0.05). Furthermore, no significant differences were recorded on fertilization rate between cows (67.5%) and heifers (63.5%) (P>0.05). Embryo quality between Nguni cows (code 1, 2.5±1.00; code 2, 1.3±0.59) and heifers (code 1, 0.8±0.41; code 2, 1.0±0.36) was similar. There was no significant difference between bull 1 (93.7%) and bull 2 (90.5%) on total sperm motility rate (P>0.05). The Super ovulatory response, fertilization rate and embryo quality between Nguni cows and heifers was similar.

Effect of Repeated Freezing and Thawing on Nguni Sperm Parameters Evaluated by Computer Assisted Sperm Analyzer System

The aims of the current study were to assess the effect of double freezing and thawing on Nguni semen parameters. Semen was collected from Nguni bulls. The Sperm Class Analyzer® system was used to evaluate sperm motility rates prior and post freezing, repeatedly. Semen samples were diluted with egg yolk citrate extender. Cooled semen samples were loaded into straws and placed inside the programmable freezer for freezing. The frozen semen straws were stored inside liquid nitrogen tank. The frozen semen straws were then thawed at 15°C (1st and 2nd freezing and thawing followed the same process) for a duration of 5 min and further assessed post-thawed at 0 and 15 min during in vitro liquid storage at 15°C. The treatment means were analyzed using Fisher’s protected t-test least. There was no significant differences recorded on raw semen sperm total motility (93.2%) and 1st frozen-thawed at 0 min (82.6%), with recovery rate of 88.6% of the sperm total motility. There was a drastic decline observed in sperm total motility during the 1st frozen-thaw at 15 min (77.6%), 2nd frozen-thaw at 0 min (31.3%) and 2nd frozen-thaw at 15 min (30.1%; P<0.05). In conclusion, the freezing and thawing process did not decrease the bull’s sperm total motility percentage during the 1st freezing and thawing process, as compared with raw semen. However, a drastic decline was observed during the 2nd freezing-thawing process, as compared with raw semen.

26 Baobab oil supplemented extender preserves post-thaw bull sperm quality parameters

The processes of semen cryopreservation and thawing affect sperm membrane integrity and motility and increases morphological defects as well as DNA damage. The most influential cause of this is oxidative stress. When endogenous antioxidant capacity of seminal plasma is reduced during the freeze-thawing process, plant extracts exhibiting strong antioxidant activity can be used as supplements for compensation. Baobab oil has gained interest because it is rich in powerful antioxidants, which could protect sperm cells from oxidative damage during cryopreservation. Our study aimed to assess the effects of baobab oil on post-thaw sperm quality parameters in an egg-yolk-based extender. Thirty semen ejaculates were collected from 15 Nguni bulls using an electro ejaculator. Semen samples were randomly allocated to control (no baobab oil), 20μL (1%), 50μL (2.5%), and 100μL (5%) baobab oil per millilitre extender. Following dilution, semen samples were loaded into 0.25-mL semen straws, equilibrated for 4h at 5°C, and transferred into a controlled rate programmable freezer. The frozen semen straws were stored in a liquid nitrogen tank (−196°C) until thawing. Semen straws were thawed (37°C/60 minutes) after 1 week of cryopreservation and analysed for (1) sperm motility using a computer-aided sperm analyser, (2) morphological defects and viability using eosin-nigrosin stain, (3) membrane integrity by hypo-osmotic swelling test, and (4) DNA integrity by terminal deoxynucleotidyl transferase dUTP nick end labelling assay. Data was analysed using analysis of variance. Treatment means were compared in relation to the control group by Dunnett's test. We found that supplementing semen extender with baobab oil at 1% significantly (P&amp;lt;0.05) preserved sperm DNA integrity (88.3±3.7) and membrane integrity (74.0±4.2) when compared with the control group (71.7±3.7 and 55.8±4.4, respectively). Baobab oil supplementation either at 1% (5.9±0.5), 2.5% (7.2±0.5), or 5% (6.0±0.5) significantly reduced sperm morphological defects compared with control (9.5±0.5). Total motility (1% (72.7%), 2.5% (72.7%), 5% (71.9%), control (59.3%)) and viability (1% (79.1%), 2.5% (79.8%), 5% (77.8%), control (67.6%)) were also improved by supplementation; however, the difference was not significant. In conclusion, it was demonstrated that supplementing bull semen extender with 1% baobab oil protects sperm from morphological defects, maintains membrane integrity, as well as preserves sperm DNA. All the baobab oil supplementation levels preserved post-thaw bull-sperm quality parameters.

21 Effect of egg yolk extracted low-density lipoprotein on cryopreserved Nguni bull semen

Egg yolk has been reported to have a beneficial effect on sperm quality following cryopreservation, and this led to its widespread use in semen extenders. However, egg yolk contains substances that inhibit respiration of sperm cells and diminish their motility rate. Moreover, it also contains low-density lipoproteins (LDL) that have a protective effect on sperm during the cryopreservation process. The objective of this study was improve cryopreservation of Nguni bull semen using egg yolk low-density protein. A total of 25 ejaculates were collected from 5 Nguni bulls aged 4 to 5 years using an electroejaculator during the natural breeding season. Collected raw semen samples were transported to the laboratory and evaluated for sperm motility, viability, plasma membrane, acrosome, and DNA integrity before dilution. Semen was randomly diluted with a sodium citrate extender supplemented with 20% egg yolk (control) and with 6, 8, 10, and 12% LDL concentrations. The diluted semen sample groups were equilibrated for 4h at 5°C. Following equilibration, semen was loaded into 0.25-mL straws and frozen in a controlled-rate programmable freezer. The groups of semen straws were then plunged into LN and transferred into LN tanks (−196°C) for storage. The frozen semen straws per treatment group were thawed at 37°C and evaluated for sperm motility, viability, plasma membrane, acrosome, and DNA integrity. Data were analysed with ANOVA using Stata V12 statistical software (StataCorp., College Station, TX), and treatment means were separated using Fisher’s protected t-test at the significant level of P&amp;lt;0.05. Sperm motility and membrane integrity were significantly higher (P&amp;lt;0.05) in frozen-thawed semen diluted with 8% LDL compared with the other concentrations. However, 6 and 8% LDL resulted in a significantly higher (P&amp;lt;0.05) live sperm, DNA, and acrosome integrity. Frozen-thawed semen diluted with 10 and 12% LDL resulted in the lowest percentages of sperm motility, live sperm, plasma membrane, acrosome, and DNA integrity following cryopreservation. In conclusion, extender containing 8% LDL resulted in improved Nguni bull semen parameters such as sperm motility, viability, plasma membrane, acrosome, and DNA integrity following cryopreservation. Further studies are required to determine the fertilizing capacity of semen diluted and frozen with LDL in vitro and in vivo.

20 Assessment of Motion and Kinematic Characteristics of Semen from Four Cattle Breeds Using Computer-Aided Sperm Analysis

Subjective semen evaluation using standard optical microscopy is the most common practice. Semen parameters routinely assessed are volume, concentration, progressive motility, and morphology. However, computer-aided sperm analysis (CASA) represents an objective evaluation, sperm assessment that are reproducible and reliable. Such semen parameters have not been evaluated in Afrikaner, Brahman, and Bonsmara bulls’ semen. The present study evaluated the sperm motion and kinematics characteristics of semen from stud Afrikaner, Brahman, Bonsmara, and Nguni bulls using CASA technology. The electro-ejaculator was used for semen collection from Afrikaner (n = 11), Brahman (n = 7), Bonsmara (n = 10) and Nguni (n = 16) bulls of known and proven fertility. Semen was collected following 4 days of resting period. The bulls ranged between 5 and 6 years of age. After collection, the semen samples were immediately transferred to a thermo-flask and maintained at 37°C for further evaluation in the mobile laboratory (Nedambale, 2014). The CASA-Sperm Class Analyzer® system (Microptic, Barcelona, Spain) was used to evaluate sperm motion, velocity, and kinematic parameters or characteristics of raw/fresh semen from 4 cattle breeds. Data were analysed using GenStat® statistical programme (VSN International, Hemel Hempstead, United Kingdom). Treatment means were compared using one-way ANOVA. The total sperm motility rate was similar for all breeds: Afrikaner (92.2 ± 4.2), Brahman (90.7 ± 9.0), Bonsmara (93.9 ± 4.0), and Nguni (96.0 ± 2.7). However, Brahman and Afrikaner bull semen had higher sperm cells moving in a progressive motility of 57.3 and 45.6%, respectively, compared with other breeds (P &lt; 0.05). Nguni, Afrikaner, and Bonsmara had the highest sperm cells moving in a rapid movement of 73.7, 72.4, and 67.4% (P &gt; 0.05), respectively. The bulls sperm trajectories had a variation, as they were recorded to be irregular and not linear (P &lt; 0.05). The straight-line sperm velocity (µm s−1), wobbling %, and amplitude of lateral head displacement % was similar for the 4 breeds (P &gt; 0.05). In conclusion, CASA technology was a useful technique for assessing differences in sperm motion and kinematic (motility and velocity characteristics) among different bull breeds.

Viability of bull semen extended with commercial semen extender and two culture media stored at 24 °C

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 °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 °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 °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 °C, and stay viable for 72 hours. Keywords : Ham’s F10, TCM-199, Triladyl, sperm motility, viability

16 COMPARATIVE STUDY ON EPIDIDYMAL SPERM TRAITS OF BONSMARA AND NGUNI BULLS

The recovery and preservation of sperm from the epididymides of slaughtered breeding bulls is a viable option in maintaining their germplasm for artificial insemination or for future use in breeding programs. The aim of this study was to evaluate and compare the quality of epididymal sperm derived from slaughtered Bonsmara and Nguni bulls. The study used 20 bulls that were 2 years of age, 10 from each breed. Twenty pairs of testes from Bonsmara and Nguni bulls were removed by orchiectomy within 20 min of slaughter, and semen was extracted from the cauda epididymis. The testicle length and weight were measured. Semen from bulls of the same breed was pooled and sperm characteristics were evaluated using a computer-aided sperm analysis (CASA). Collected semen samples were then extended (1:1) in egg yolk citrate (EYC) extender and stored at 5°C. Sperm motility rate was evaluated at 0, 24, 30, and 48 h during in vitro semen storage. Data were analysed by ANOVA. The Bonsmara bull’s testicle length (13 ± 0.31cm) and weight (290 ± 0.24 g) were higher than that of the Nguni bulls (10.3 ± 0.11 cm and 275 ± 0.42 g; P &lt; 0.05). However, there were no significant differences in fresh total sperm motility rate (74.4 ± 2.14 v. 75.8 ± 3.11%) or concentration (5.31 ± 1.14 v. 5.83 ± 2.09 × 109) of Bonsmara and Nguni bulls, respectively. There were no significant differences observed for total sperm motility at 24 h (49.3 ± 1.18 v. 52.6 ± 2.60%) and 30 h (40.5 ± 1.94 v. 43.4 ± 2.81%) between the Bonsmara and Nguni bulls, respectively. The total sperm motility at 40 h was &lt;20% for both Bonsmara and Nguni bulls on the EYC extender. There were no differences in sperm motility rate of the epididymal semen of Bonsmara and Nguni bulls. We concluded that it is possible to derive semen from the epididymis of indigenous Nguni and composite Bonsmara bulls.

15 QUANTIFICATION OF BULL SPERM TRAITS AS ASSESSED BY COMPUTER-ASSISTED SEMEN ANALYSIS AND THE RELATIONSHIP TO PREGNANCY RATE FOLLOWING CONTROLLED BREEDING

The bull’s contribution through artificial insemination to reproductive efficiency is of great biological importance. The objectives were (1) to compare the oestrous synchronization response of Bonsmara and Nguni cows; and (2) to find the relationship between cow’s conception rate (in vivo and in vitro fertilization) and bull sperm motility rate assessed by computer-assisted semen analysis (CASA) following AI. For the in vivo sperm fertility test, 100 Bonsmara and 482 Nguni cows were randomly selected and subjected to oestrous synchronization protocol and AI with frozen–thawed assessed semen by CASA before AI. Briefly at Day 0, cows were inserted with an intravaginal CIDR® (1.9 g), which was removed on Day 7. Prostaglandin was then administered (2 mL) on Day 8 and a heatmount detector was placed on the hindquarter of each cow. For the in vitro sperm fertility test, collected oocytes from slaughterhouse were in vitro matured (n = 360) and in vitro fertilized (sperm/mL) in 100-µL droplets (final volume) of BO-IVF medium per treatment bulls (Bonsmara or Nguni bull). The frozen/thawed semen straws of Bonsmara and Nguni bulls were randomly selected and used under the same IVF conditions. The thawed bull’s sperm characteristics were examined by CASA before in vitro fertilization. Data were analysed using ANOVA. Treatment means were compared using the Fisher’s protected least significant difference t-test. There was no significant difference in oestrous response for the Bonsmara (83.0%) and Nguni (90.8%) cows, respectively. The Bonsmara cows recorded a significantly higher pregnancy rate (59.0%) compared with the Nguni (37.1%) cows (P &lt; 0.05). Sperm traits such as total motility (TM), progressive motility and rapid were found to be positively correlated with conception rate (r = 0.06, 0.03, and 0.08, respectively; P &lt; 0.01), although correlations were low. There was no difference in the average frozen–thawed sperm TM rate of Nguni (92.2%) and Bonsmara (81.0%). There was a lower fertilization rate following IVF with Bonsmara and Nguni bull sperm. In conclusion, Nguni cows had similar oestrous response as Bonsmara cows. The sperm traits from Bonsmara and Nguni bulls were found to be related to in vivo conception and in vitro fertilization rate when sperm cells were assessed by CASA technology. However, the pregnancy rate was lower in Nguni cows.

163 EVALUATION OF VIABILITY OF BULL SEMEN COLLECTED BY ELECTRO-EJACULATION USING COMMERCIAL SEMEN EXTENDER AND 2 CULTURE MEDIA AT CONTROLLED ROOM TEMPERATURE

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 &gt;70% and morphological defects &lt;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 &lt; 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 &gt; 0.05), in viability rate between Ham’s F10 (26.5%) and TCM-199 (25.0%). No significant difference (P &gt; 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 &gt; 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.

37 DOUBLE FREEZING AND THAWING OF NGUNI BULL SEMEN

Indigenous bulls semen are important for conservation programs. The objectives of this study were to evaluate the effects of repeated freezing and thawing on sperm motility characteristics. Semen was collected from 4 Nguni bulls by means of electro ejaculator and stored in a thermo flask (37°C). Sperm total motility, progressive and nonprogressive motility, and velocity were assessed using computer-aided sperm analysis before and after freezing. Semen was then diluted with egg yolk citrate extender (fraction A), then followed by 12% of glycerol + egg yolk citrate extender (fraction B, Seshoka et al. 2012). Diluted semen samples were equilibrated for 4 h at 5°C. After the equilibration period, samples were loaded into 0.25-mL straws and transferred into a controlled rate programmable freezer. After the target temperature of –130°C was reached, semen straws were stored in a LN tank (–196°C). After 3 months of storage, straws were thawed at 15°C (first and second freezing and thawing followed the same process) for 5 min and further evaluated post-thawed at 0 and 15 min during incubation at 15°C. Treatment means were separated using Fisher’s protected t-test least. No significant differences were recorded between the raw semen total sperm motility percentage (93.2%) and first frozen-thawed at 0 min (82.6%), with the total sperm motility rate recovery of 88.6%. In addition, there was a marked decline recorded in sperm total motility during the first frozen-thawed at 15 min (77.6%), second frozen-thawed at 0 min (31.3%), and second frozen-thawed at 15 min (30.1%; P &lt; 0.05). The sperm curvilinear velocity and average path velocity was reduced following first frozen-thawed (P &lt; 0.05) but remained constant and stable between the treatment groups (P &gt; 0.05). In conclusion, the freezing-thawing process did not reduce the Nguni bull total sperm motility during the first freezing and thawing process, compared with raw semen. However, a drastic decline was recorded during the second freezing-thawing processes, compared with raw semen.

38 QUAIL EGG YOLK IN CITRATE EXTENDER IS SUITABLE FOR CRYOPRESERVATION OF NGUNI BULL SEMEN

Traditionally, commercial hen egg yolk has been used in extenders or freezing media because of its easy availability. However, the use of quail egg yolk has not been used for preserving Nguni bull semen. The aim of the study was to compare the suitability of different quail egg yolk concentrations (5, 10, 15, and 20%) for cryopreserving Nguni bull semen. Semen was collected from 14 stud Nguni bulls with the aid of electro ejaculator. Collected semen samples were kept in a thermos-flask containing warm water at a temperature of 37°C and transported to the laboratory for further analyses. The sperm motility traits were evaluated using computer-aided sperm analysis prior extension. Semen samples were then randomly allocated into 5, 10, 15, and 20% of quail egg yolk and 20% concentration of chicken egg yolk (control) in citrate extender. The extender was supplemented with 12% of glycerol (Seshoka et al. 2012) as a cryoprotectant, and semen samples were diluted (1 : 2) and equilibrated for a period of 4 h at 5°C. After equilibration, semen samples were loaded into 0.25-mL straws, placed into a controlled rate programmable freezer, and stored in a LN tank (–196°C) until thawing. Frozen semen straws were thawed in a water bath at 37°C for 1 min. Thawed semen was evaluated for sperm motility traits using a computer-aided sperm analysis system. Data were analysed with ANOVA. A significant difference was recorded between fresh total sperm motility rate (99%) and frozen-thawed semen samples with either 5% (87.3%) quail or 20% (87.6%) chicken egg yolk citrate extender compared with 10% (92.6%), 15% (91.2%), or 20% (89.9%) quail egg yolk citrate extender. Moreover, fresh semen also resulted in a significantly higher progressive sperm motility rate (39.3%) as compared with frozen-thawed with 5% (26.2%) or 20% quail (28.5%) or 20% chicken (22.7%) egg yolk citrate extender. The results also demonstrated that the use of 10, 15, and 20% quail egg yolk in citrate extender yielded comparable results on total sperm motility with fresh semen as compared with 5% quail and 20% chicken egg yolk. In conclusion, quail egg yolk extender provided sufficient cryo-effectiveness to cryopreservation of Nguni bull semen.

349 SUPEROVULATORY RESPONSE AND EMBRYO QUALITY RECOVERED FOLLOWING FLUSHING NGUNI HEIFERS AND COWS

Most of the South African indigenous cattle breeds are facing genetic degradation due to unselective crossbreeding and irregular matings especially in small scale farming. The aims of the study were to compare superovulatory (SO) response, fertilization rate and to evaluate embryo quality recovered from superovulated Nguni stud cows and heifers. Nguni stud cows (n = 15) and heifers (n = 10) aged 4–6 and 2 years, respectively, were used as embryo donors for ex situ conservation. Nine days following heat observation for the first oestrus synchronisation protocol, superstimulation of donor cows and heifers were administered with a total of 350 mg of FSH (Follotropin-V®, porcine pituitary glands extract) divided into 2 injections daily 12 h apart (6 a.m. and 6 p.m.) for 4.5 days on a decreasing dosage. Two injections of prostaglandin F2α were administered on Day 3 and 4 of FSH injections. Semen was collected from 2 Nguni bulls (bull 1 and 2) and assessed by computer aided sperm analysis before artificial insemination (AI). The AI was conducted with fresh semen 3 times, 12 h apart, and the first AI was performed at the onset of oestrus. Semen from bull 1 was used to inseminate the cows, and bull 2 was used for heifers. Embryos were flushed 7 days after AI using a nonsurgical technique. Embryos were evaluated under stereo microscope and classified according to IETS standard codes (code 1, good and excellent; code 2, fair). Recovered embryos were then vitrified and stored for future use. Superovulatory response was measured by counting number of corpus lutea in each ovary. Data were analysed using 1-way ANOVA (SAS, 2003, SAS Institute Inc., Cary, NC, USA). There was no statistical difference between bull 1 (93.7%) and bull 2 (83.5%) on total sperm motility rate. Furthermore, no significant differences were recorded on fertilization rate between cows (67.5%) and heifers (53.5%). There was also no significant difference on the proportion of Nguni cows (40%) and heifers (40%) that responded to superovulation treatment. There was a significant difference on the ovary reaction (number of corpus lutea) of cows (11.3 ± 1.41) and heifers (4.0 ± 0.57). There were no significant differences recorded on the embryo quality recovered between Nguni cows (code 1, 2.5 ± 1.00; code 2, 1.3 ± 0.59) and heifers (code 1, 0.8 ± 0.41; code 2, 1.0 ± 0.36). However, cows had higher numbers of unfertilized ova than heifers (5.5 ± 1.05 and 1.8 ± 0.47, respectively) and degenerate embryos (3.7 ± 1.00 and 1.3 ± 0.39, respectively). Although superovulatory response of both Nguni cows and heifers was low, Nguni cows had higher ovarian response than heifers. Moreover, the quality of embryos recovered was similar for both Nguni cows and heifers.

53 COMPARISON OF 4 DIFFERENT CRYOPROTECTANTS ON FREEZING NGUNI BULL SPERMATOZOA EVALUATED BY COMPUTER-AIDED SPERM ANALYSIS

Cryopreservation has been reported to damage approximately 40 to 50% of viable spermatozoa in bulls. It is critical to evaluate frozen-thawed spermatozoa with computer-aided sperm analysis (CASA) and find a suitable cryoprotectant for Nguni semen. The study was conducted to compare cryo-effectiveness of glycerol (GLY), dimethyl sulfoxide (DMSO), propanediol (PND), and ethylene glycol (EG) cryoprotectants at 12% concentrations during freezing of Nguni bull semen. Semen was collected from 18 stud Nguni bulls of proven fertility with the use of an electro-ejaculator. Collected semen samples were kept in a thermo flask (37°C) and transported to the laboratories. Semen samples were pooled and sperm motility rate was evaluated using CASA. Semen was then diluted (1 : 2; vol : vol) with egg-yolk citrate extender supplemented with either 12% GLY, DMSO, PND, or EG. Semen samples were equilibrated for 4 h at 5°C. After equilibration, samples were loaded into 0.25-mL straws and placed into the controlled rate programmable freezer using a customized freezing curve (from 5 to –5°C at 0.008°C min–1 and from –3 to – 130°C at 6°C min–1). Following thawing of semen, artificial insemination was conducted on 104 oestrus-synchronised Nguni cows. The IVF was also conducted on 120 oocytes to check the cleavage rate. Data were analysed using ANOVA. There was a significant difference (P &lt; 0.05) between raw total sperm motility (94.70 ± 2.63) and frozen-thawed sperm total motility with GLY (77.80 ± 11.03), EG (20.35 ± 11.86), DMSO (15.68 ± 10.14), and PND (11.19 ± 11.27) groups. The pregnancy rate following artificial insemination was 75.9% and a total of 86.6% oocytes had cleaved after fertilization with frozen (12% GLY)/thawed semen. In conclusion, cryopreservation process reduced sperm motility and velocity rates, regardless of cryoprotectant. Egg-yolk citrate extender supplemented with 12% glycerol had recorded the highest post-thaw sperm motility rate.

Determination of economic weights for breeding traits in indigenous Nguni cattle under in-situ conservation

This study was conducted to determine the economic weights of most preferred traits in young breeding Nguni bulls and first parity cows. Fifty-four farmers from low-input cattle production enterprises participated in the choice experiment. Sixteen individual animal profiles were formulated from four traits of three levels each using a fractional orthogonal design of SPSS 14.0 (2005). 120 pair-wise choices were deduced for each breeding animal class. A total of 6480 (54×120) observations were obtained for each class of the animals. Data was subjected to multinomial logit (MNL) models using econometric software NLOGIT 4.0.1 Version (2007). All computed economic values for bull traits were significant (p<0.05). The economic weights of bull traits were poor body condition score (−0.99±0.095), good body condition score (0.45±0.073), over-conditioned (base level), low tick infestation (0.57±0.103), medium tick infestation (0.58±0.084), high tick infestation (base level), high aggression and mating behavior (4.41±0.095), average aggression and mating behavior (2.53±0.094), and low aggression and mating behavior (base level). The economic weights of first parity cow traits were poor body condition score (−0.06±0.055), good body condition score (1.08±0.061), over-conditioned (base level), low tick infestation (1.50±0.059), medium tick infestation (0.83±0.067), high tick infestation (base level), age at first calving of ≤27 months (2.37±0.068), age at first calving of 27–36 months (1.30±0.076), and age at first calving of >36 months (base level). Farmers were willing to pay R37,939 (US$4864) for a bull with high aggression and mating behavior score and R17,185 (US$2203) for a first parity cow of less than 27 months old. Enterprise ownership and demographics factors of the farmers were significant in determining economic weights within trait levels. Economic weights were high for reproductive efficiency of the breeding animals followed by the high adaptive characteristics. The choice experiment procedure can be the tool for determining importance of animal characteristics under low-input production systems. It is recommended to make use of the economic weights of preferential traits in designing selection models.

76 THE EFFECT OF GLYCEROL CONCENTRATIONS IN EGG-YOLK CITRATE EXTENDER ON THE QUALITY OF CRYOPRESERVED NGUNI BULL SEMEN

There are bull shortages in South African poor rural areas. Artificial-insemination technology could play a significant role on breeding emerging farmer’s cattle. The objective of this study was to compare glycerol concentrations (0, 4, 8, or 12%) during freezing of Nguni bull semen to conduct AI in different villages. Semen was collected by electro ejaculator from 2 Nguni bulls of known and proven fertility. Collected semen samples were kept in a thermo flask (37°C) and transported to the laboratories within 10 min after collections. Semen samples were pooled and evaluated by Sperm Class Analyser® and allocated randomly per treatment. Semen was then diluted (1 : 2 v:v) with egg-yolk citrate extender supplemented with either 0% (negative control), 4, 8, and 12% of glycerol concentration or AndroMed® (positive control). Semen samples were equilibrated for 4 h at 5°C. After equilibration period, samples were loaded into 0.5-mL straws and placed horizontally into the controlled rate (–5, –8, –10, –12, –15, –25, –35°C min–1) from 5°C until target temperature of –80°C is reached. The frozen semen straws were stored in a liquid nitrogen tank (–196°C) until thawing. Treatment means were separated using Fisher’s protected t-test least, and data are presented as mean ± SD. There was a significant differences (P &lt; 0.05) between raw total sperm motility (83.3 ± 19.3) and frozen–thawed sperm with either 0% (0.0 ± 0.0), 4% (30.2 ± 13.4), 8% (47.9 ± 12.5), or 12% (61.5 ± 4.7) of glycerol and on AndroMed® (27.7 ± 17.8) group. Regardless of the glycerol concentrations used, the freezing-thawing process reduced (P &lt; 0.05) the Nguni total sperm motility rate compared to uncryopreserved sperm (83.3 ± 19.3). In conclusion, egg-yolk citrate extender supplemented with 12% glycerol yielded a better (P &lt; 0.05) total sperm motility rate (61.5 ± 4.7) as compared with the 0% (0.0 ± 0.0), 4% (30.2 ± 13.4), 8% (47.9 ± 12.5), and AndroMed® (27.7 ± 17.8) group. Further studies are required to test other levels of glycerol concentrations (&gt;12%) on freezing Nguni semen and conducting AI.

107 MOTILITY PARAMETER PATTERNS OF NGUNI BULLS: EFFECT OF VARIOUS GYCEROL CONCENTRATIONS FOLLOWING CRYOPRESERVATION

The Nguni breed of South Africa is small, hardy, disease-tolerant, thrives on poor pasture, and was regarded as an inferior breed in the past. For optimizing routine fresh and freezing of Nguni bull semen analysis, 3 different concentrations of glycerol (7, 10, and 14%) were examined. Ten ejaculates were collected from each 6 Nguni bulls using electro-ejaculator at ARC, Irene, South Africa. Following semen collection, semen was examined for macroscopic (volume, pH, and concentration) and microscopic (motility) parameters. The semen was extended with Tris + 10% egg yolk diluent at a ratio of 1 : 2 (v/v) and frozen at different concentrations of glycerol (7, 10, and 14%). The semen was then evaluated using the sperm class analyzer (SCA; CASA system) for progressive motility parameters. Fresh and frozen-thawed were fixed and stained with Nigrosin-Eosin for morphology (dead and live). Data were analyzed by ANOVA. There was a significant difference among individual Nguni bull spermatozoa volume and concentration. Analyzed frozen-thawed Nguni spermatozoa resulted in a significant (P &lt; 0.05) difference of spermatozoa motility parameters frozen in 10% glycerol (68%) compared with 7 (41%) and 14% glycerol (30%). In conclusion, Nguni spermatozoa can be cryopreserved successfully when 10% of glycerol concentration is used. The results of this study will improve the viability of cryopreserved Nguni bull spermatozoa following the development of a South African semen cryo-gene bank. This study was supported by grants from National Research Foundation (NRF), Hungarian, South African Bilateral Scientific and Technological (TETNo. OMFB-00302/2008, RT24000) collaborative project. Department of Agriculture Forestry and Fisheries (DAFF, RPPP15).

Heritable testicular hypoplasia in Nguni (Bos indicus) bulls: vascular characteristics and testosterone production.

The biased unilateral occurrence of heritable gonadal hypoplasia was investigated by examining the gross- and microanatomy of the testicular artery and vein, testicular blood flow and testicular testosterone secretion in normal Nguni bulls and in Nguni bulls showing unilateral left, unilateral right and bilateral hypoplasia of the testis. A high incidence of branching of the testicular artery was found ipsilateral to hypoplastic testes. The branching occurs a short distance from the dorsal aorta: one branch proceeds to the testis, the other to the ipsilateral kidney. The association between arterial branching to the kidney and ipsilateral hypoplasia of the testis held for both unilaterally left and unilaterally right hypoplastic bulls. Variations in the anatomy of the testicular vein occurred in both normal and hypoplastic bulls but there was no specific association between the variations and ipsilateral hypoplasia. The lumen diameter of the testicular artery or branch correlated with testis mass. Wall thickness of the artery ipsilateral to hypoplastic testes was not different from that in normal bulls, discounting hyperplasia of the endothelium. Total blood flow to the testis correlated with testis mass. The secretion rate of testosterone from hypoplastic testes was lower than that of normal testes but there was no difference when compared on a unit mass basis.

The beef tenderness model

In Phase1 of this study, three breed types (Simmentaler-, Brahman- and Nguni bulls; n = 60 each) were grain-fed and slaughtered at 12 months of age (A-age, fat-class 2). Feed was withdrawn for either three hours or 24 hours pre-slaughter. Within each feed withdrawal group, three electrical stimulation (ES) treatments were applied, viz. ES for 15 seconds, 120 seconds or no stimulation. In Phase 2, the effects of animal age and feeding regime were investigated using of A-age (feedlot and pasture), AB-age (feedlot and pasture) and B-age (pasture) animals. All carcasses were electrically stimulated for 15 seconds. Longer feed withdrawal increased dark-firm-dry (DFD) meat occurrence (pHu &gt;6) in the Nguni and Simmentaler-cross. Brahman-cross longissimus (LL) tended to be more tender with paler colour and higher drip loss when 120 ES was applied. Longer feed withdrawal recorded higher Warner Bratzler shear force (WBSF) than three hour feed withdrawal. However, ES neutralized the effect of stress on tenderness. On average the AB-age feedlot animals produced the most tender LL followed by the B-age pasture and A-age feedlot which was similar to the AB-age pasture. A-age pasture animals produced the least tender LL steaks. The calpain proteolytic system played a pivotal role in determining the ultimate meat tenderness and although connective tissue becomes less soluble in older animals it did not play the determinant role in tenderness in this study. The intramuscular fat (&lt; 3%) played an important role in the tenderness outcome of Nguni LL which marbled well at AB-age.Keywords: Meat tenderness, pH and temperature decline profile, pre- and post-slaughter conditions, production systems