Yolk Citrate Extender Research Articles

Coconut water combined with purebred chicken egg yolk as an alternative semen extender for Madura Pote bucks semen preservation

One of the many efforts to increase the quality of livestock genetics is through artificial insemination (AI). Other than increasing it, AI can be conducted to preserve semen. A successful AI is determined by seminal quality, therefore, a method to preserve semen for a longer storage time is needed. The method used is adding an extender that fulfills prerequisites for a semen extender such as coconut water combined with egg yolk citrate extender. Coconut water is rich in carbohydrates, protein, vitamins, and antioxidants while egg yolk contains lecithin. This study aims to find out the Pote buck spermatozoa quality stored in coconut water and egg yolk extender. This study uses three groups of treatments (T0: 0.1 ml semen + 0.9 ml egg yolk citrate, T1: 0.1 ml semen + 0.9 ml coconut water, and T2: 0.1 ml semen + egg yolk citrate (20%) + coconut water). All three of these are stored at 5oC and evaluated every day until day 5 of their motility, viability, intact plasma membrane, abnormality, and MDA level. Data analysis used is ANOVA and a further test called BNT is conducted if a significant difference is determined. No significant difference was found between T0 and T1 (p>0.05). The highest progressive motility, viability, and intact plasma membrane (%) among the three groups of treatments happened to be from T2. Meanwhile, a low percentage of spermatozoa abnormality and MDA level were also found in T2 with its extender being coconut water combined with egg yolk citrate. To conclude, the best extender for storing Pote buck semen is stored at 5oC is coconut water combined with egg yolk citrate extender.

Effect of the addition of soursop juice (Annona muricata L., 1753) to egg yolk citrate extender on the quality of bull semen

The present study was carried out to investigate the effect of the addition of soursop (Annona muricata L., 1753) juice to the citrate-egg yolk extender (EYC) on the quality of cryopreserved bull semen. After pre-evaluation, the semen was distributed in five extenders T1, T2, T3, T4 and T5 containing respectively 0% soursop juice (SJ) (T1), 25% SJ (T2), 50% SJ (T3), 75% SJ (T4), 100% SJ (T5) and microscopic evaluation was performed daily to assess plasma membrane integrity (PMI), progressive motility of spermatozoa (MP) and sperm pH until all sperm were dead. The results indicate a significant increase in spermatozoa PMI and MP (p<0.05) in the case of treatment with 50% SJ (T3). Spermatozoa showed a motility of 50.78% 9 days later in the citrate-egg yolk extender containing 50% SJ (T3), whereas at day 9, spermatozoa in the other extenders had a progressive motility of less than 50%. Similar results were obtained for PMI with 50.78% of spermatozoa having an intact plasma membrane 9 days later in the citrate-egg yolk extender containing 50% SJ whereas the PMI of cells in the other stents was less than 50%. However, the pH of the extender containing 50% SJ was closest to 7 during the experimental period. The results of this experiment suggest that the addition of 50% soursop juice (T3) to the citrate-egg yolk extender could be adopted as an acceptable alternative to traditional egg yolk-based extenders for the cryopreservation of bull semen.

Effect of black cumin (Nigella sativa) seeds extract in tris egg yolk citrate extender on sperm motility of cryopreserved Surti buck semen

Effect of black cumin (Nigella sativa) seeds extract in tris egg yolk citrate extender on sperm motility of cryopreserved Surti buck semen

Effect of black cumin (Nigella sativa) seeds extract in tris egg yolk citrate extender on viability of cryopreserved Surti buck semen

Effect of black cumin (Nigella sativa) seeds extract in tris egg yolk citrate extender on viability of cryopreserved Surti buck semen

Comparison of different poultry egg yolks-citrate extender with green tea (Camellia sinensis) extract addition on Sapudi ram spermatozoa quality in chilled temperature storage

Preservation of ram semen has an impact on the exposure of artificial conditions to the spermatozoa which was followed by overproduction of reactive oxygen species (ROS) and resulted in functional damages of spermatozoa. This study aimed to determine the effect of the combination of chicken, quail, and duck egg yolks with green tea extract (GTE) on the quality of Sapudi ram spermatozoa at 5°C storage. Ejaculates of Sapudi ram with more than 70% spermatozoa motility were divided into four groups. Semen were extended 1 : 10 in citrated-chicken egg yolk extender (control group, T0), and citrated yolk of chicken (T1), quail (T2), and duck (T3) egg added with 0.05 mg GTE per 100 mL of extender. Extended semen was kept in a tube, which was put in a beaker containing distilled water and stored in a refrigerator (5°C). Spermatozoa motility, viability, and plasma membrane integrity (PMI) were evaluated daily for five days. The results showed that GTE in duck egg yolk citrate extender could maintain the highest spermatozoa motility, viability, and PMI for five days (p <0.05). It could be concluded that the duck egg yolk citrate extender with the addition of GTE (Camellia sinensis) was beneficial for maintaining the semen of Sapudi ram spermatozoa at chilled temperatures. Further study is needed to use the extender for freezing Sapudi ram semen.

The effects of green synthesized anionic cupric oxide nanoparticles on Zaraibi goat spermatozoa during cryopreservation with and without removal of seminal plasma

Sperm motility, normal morphology, viability, spermatozoa DNA damage, and lipid peroxidation are all affected by semen cryopreservation. The goal of this study was to see how effective cupric oxide nanoparticles (CuONPs) are as a cryo-extender additive on post-thawed sperm parameters. An artificial vagina was used to collect semen samples from five mature Zaraibi bucks (2–3 years). Ejaculates were pooled and separated into two fractions (A&B), a fraction (A) was left without being centrifuged and a fraction (B) was centrifuged to remove seminal plasma. Both fractions were diluted with tris egg yolk citrate extender (TECE) and then divided into five equal aliquots, each supplemented with (0, 10, 20, 40, and 60 ppm/ml) CuONPs. The findings revealed that removing seminal plasma before cryopreservation harms sperm parameters. Sperm motility, viability index, membrane integrity, biochemical antioxidant marker, DNA integrity, and MDA level improved after supplementation with CuONPs up to 60 ppm/ml, the most prominent significant positive effect was obtained with the highest dose (60 ppm/ml) without removal of the seminal plasm compared to control group. In conclusion: The presence of seminal plasma with a high concentration of CuONPs (up to 60 ppm/ml) may help to mitigate the negative effects of cryo-preservation.

​Evaluation of Antioxidant Potential of Taurine in Tris Egg Yolk Citrate Extender in Surti Buck Semen Preserved at Refrigerated Temperature

Background: Taurine supplementation in tris egg yolk citrate (TEYC) extender can improve antioxidant defense and reduce motility degeneration rate in semen of Surti buck preserved at refrigeration temperature. Present study has evaluated antioxidant effect of different concentrations of taurine in TEYC extender on oxidative stress, antioxidant defense and motility degeneration rate in Surti buck semen preserved at refrigerated temperature. Methods: Four Surti bucks (age greater than 2years) were selected. Total 64 ejaculates (16 per buck) were collected during a period of 8 week. Post-collection they were pooled and stored at refrigeration temperature after dividing into 5 groups based on different taurine concentrations in TEYC extender viz. 0 mM (control T1), 25 mM (T2), 50 mM (T3), 75 mM (T4) and 100 mM (T5) taurine maintaining final concentration of 200 x 106 sperm/ml (pH 6.5-6.8). Evaluation of motility degeneration rate at 24, 36 and 48 hours and glutathione as well as lipid peroxidation (MDA) at 0 and 48 hours was done. Result: Supplementation of taurine @50 mM in TEYC extender caused post-chilling significant increase in reduced glutathione (GSH) levels, lowering of lipid peroxidation (in terms of MDA production) and reduction of motility degeneration rate (MDR)% in semen of Surti bucks. It improved antioxidant defense thereby maintaining good quality of semen.

Effect of taurine in tris egg yolk citrate extender on quality of surti buck semen at refrigerated temperature

Semen was collected from four Surti bucks (>24 months-old) twice a week for 8 weeks (16 ejaculates per buck) during November, 2020 to March, 2021. Ejaculates of all four bucks were pooled and divided into five equal aliquots. The aliquots were diluted with tris-egg yolk-citrate (TEYC) extender containing 0 (control T1), 25 (T2), 50 (T3), 75 (T4) and 100 mM (T5) taurine with final concentration of 200x106 sperm/ml (pH 6.5 to 6.8) in each. At 24, 36 and 48 h of refrigerated storage, individual sperm motility, per cent live sperm and HOST reacted sperm were significantly (p<0.01) higher in T3 (50 mM) and abnormal sperm % was significantly (p<0.01) lower in T3 (50 mM) and T4 (75 mM) as compared to other groups. With increasing duration of storage, significant (p<0.01) increasing trend for abnormal sperm per cent and decreasing trend for rest of the characters were observed. Motility showed significant (p<0.01) positive correlation with live spermatozoa, abnormal spermatozoa and HOST-reacted spermatozoa. Live count showed significant (p<0.01) negative correlation with abnormal spermatozoa and significant (p<0.01) positive correlation with HOST-reacted spermatozoa. Live spermatozoa showed significant (p<0.01) negative correlation with abnormal spermatozoa and HOST non-reacted spermatozoa. Abnormal spermatozoa showed significant (p<0.01) negative correlation with HOST-reacted spermatozoa. It was concluded that supplementation of 50 mM taurine in TEYC extender maintained optimum quality of buck semen at refrigerated temperature up to 48 h.

English

Using a tabular design, the bacteriological profile of egg yolk forms and different formulations of yolk-citrate semen extenders across different storage periods was examined. Twenty-one eggs less than 24 h old were randomly selected from 3 crates of eggs. The yolks were harvested and divided into two equal parts. One part was centrifuged for 1 h at 3500 revolutions per minute (rpm) to get clarified yolk. The remaining part was left whole. Each part was divided into three aliquots, one of which was cultured immediately (pre-storage) on&nbsp; MacConkey, Chocolate, Deoxycholate Citrate and Sabouraud Dextrose Agars and the other two stored each for 24 and 48 h before culture on the same media. The cultures were incubated for 24 h at 37&deg;C. Left over egg yolks were pooled according to forms and kept frozen (-20&deg;C) before being used to formulate extenders. Four different extenders were prepared using the clarified and whole yolks with or without antibiotics. The extenders were treated as previously described for egg yolk. Plates with apparent colonies were subjected to biochemical tests to identify the isolates and antibiotic susceptibility testing after the colonies were counted. Clarified yolk was less contaminated compared to whole egg yolk. Staphylococcus aureus was isolated from whole egg yolk and it was sensitive to Streptomycin, Amoxicillin, Neomycin, Penicillin and Cefoxitin but resistant to Gentamicin. Extenders with antibiotics were less contaminated than extenders without antibiotics. Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli and Salmonella species were isolated from semen extenders without antibiotics. All isolates were sensitive to Gentamicin. In conclusion, clarified yolk and extenders containing clarified yolk with antibiotics had no contamination. Hence, clarified yolk should be used in the preparation of extenders instead of whole yolks. Also, Gentamicin should be included in extender formulations to reduce bacterial contamination. Key words: Egg yolk, whole yolk, clarified yolk, bacterial culture, yolk-citrate extender, antibiotics.

Effect of extenders, cryoprotectants, and freezing methods on sperm quality and fertilisation rate of the European sea bass (Dicentrarchus labrax)

Fish semen cryopreservation is a valuable technique to sustain good quality sperm for fertilization, facilitating threatened species conservation and broodstock genetic development. The present investigation aimed to develop suitable standardized protocols to cryopreserve sea bass (Dicentrarchus labrax) semen. Thus, four experiments were conducted to determine the most effective extender (egg-yolk citrate, urea-egg-yolk, and 0.9% NaCl), cryoprotectant (DMSO, glycerol, ethylene glycol “EG”, and methanol), sperm/cryodiluent dilution ratio (1:2, 1:4, 1:10, and 1:20) and equilibration time (0, 5, 15, or 30 min) to maintain the sperm content after cryopreservation. The consistency of post-thaw semen was assessed, with motility percentage, motility duration, and fertilization rate all being taken into account. When sperms were thawed, the consistency was at its best, especially when sperm was diluted with a ratio of 1:20 with egg-yolk citrate extender containing 10% DMSO, with a 15-min equilibration period before cryopreservation. Sea bass sperm cryopreserved using this protocol exhibited fertilisation rates, class motility, and motility duration close to fresh semen. Thus, the cryopreservation method developed in the present investigation proved to be an efficient and effective approach to augment the cryosurvival of sea bass spermatozoa.

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.

Improvement of Semen Quality in Rams by Adding Some Biostimulants During Cooling

This study was carried-out to throw the light on the effect of some biostimulants as L-carnitine; 0.1mg/ml , Omega 3; 10 ng/ml and moringa olifera; 100 μg/ml on sperm progressive motility and livability percentages, abnormalities, acrosomal integrity of rams semen cooled in refrigerator (+ 5) during different periods (0,24,48,72,96 hrs) in different extender [Egg yolk citrate extender 10% egg yolk (EG) (1st dil) , tris based extender 2.5% EG (2nd dil), tris based extender 10% EG (3rd dil) ]. The results indicated that, the best treatment group that improved the sperm abnormalities and acrosomal integrity % observed in L-carnitine treated group followed Omega3-treated group and the least treatment observed in Moringa olifera treated group. The best treatment improved the progressive sperm motility and livability percentage of sperm cells observed in the groups treated with Omega-3, followed moringa olifera and the least observed in L-carnitine treated group, also the best diluent is tris based extender 10% egg yolk (3rd) the results also cleared that, by increasing the time of cooling there is a decrease of sperm quality and its ability to conception. It could be concluded that, the biostimulants used in this study specially Omega-3, and moringa olifera in tris based extender 10% egg yolk (3rd) improved semen quality of rams preserved at refrigerator for 4 days specially

Effect of Simmental bull seminal plasma protein in egg yolk-citrate extender on Kacang buck semen fertility

The genetic resources of Indonesia's indigenous Kacang goat require preservation. Artificial insemination is expected to accelerate population increases and preserve genetic resources simultaneously. The present study was the maiden attempt for cryopreservation of Kacang buck sperm. The objectives of this study were to determine whether the supplementation of superior Simmental bull seminal plasma protein in egg yolk-citrate extender could improve the quality of post-thawed Kacang buck sperm, increase conceptions rates, and improve kidding rates. Buck semen was diluted without supplementation (T0) and with supplementation of 2.5 mg (T1) and 5 mg (T2) of Simmental bull seminal plasma protein per mL egg yolk-citrate extender. Extended semen was packed in 0.25 mL straw as cryopreserved frozen semen. Post-thawed semen samples were evaluated for viability, motility, intact plasma membranes, malondialdehyde level, and DNA fragmentation. Estrus was synchronized for sixty Kacang does, which were divided randomly into three groups and inseminated using post-thawed semen. The progesterone serum concentration of the does was measured 7 and 22 days post-insemination to detect ovulation and conception. Pregnancy was confirmed using abdominal palpation at 43 days post-insemination and by observing birth. The T1 group showed the highest (P < 0.05) post-thawed viability, motility, and intact plasma membrane. Conception, pregnancy and kidding rates were also higher in T1 than other treatment groups. In conclusion, the 2.5 mg Simmental bull seminal plasma protein supplementation per mL egg yolk-citrate extender provided the best seminal quality and fertility of post-thawed Kacang buck semen.

Effect of coconut water in tris egg yolk citrate extender on cauda epididymal buck spermatozoa motility preserved at refrigeration temperature

Effect of coconut water in tris egg yolk citrate extender on cauda epididymal buck spermatozoa motility preserved at refrigeration temperature

Anti-Oxidant Effect of Quercetin in Tris Egg Yolk Citrate Extender on Surti Buck Semen Preserved at Refrigerated Temperature

Anti-Oxidant Effect of Quercetin in Tris Egg Yolk Citrate Extender on Surti Buck Semen Preserved at Refrigerated Temperature

Effect of Tomato Juice in Tris-Yolk-Citrate Extender onRefrigeration Preservation of Cauda Epididymal Spermatozoa of Buck

The study was carried out on the preservation of epididymal spermatozoa of buck at refrigerated temperature without and with tomato juice as a supplement in Tris egg yolk citrate extender. The eight pairs of testicles including epididymis (total 16) from slaughtered bucks were collected within 2–4 hours of their slaughter. Sperms collected from cauda epididymis were preserved at refrigerated temperature up to 48 hours in tris egg yolk citrate extender at 300 million sperm/mL with different concentration of tomato juice (0%, 4%, 6%, 8%, and 10%) and the physical characteristics of spermatozoa were assessed to know the effect of tomato juice (Tj). The mean dead, abnormal and HOST non-reacted spermatozoa increased significantly (p less than0.05) at every 12-hour intervals of preservation in the dilutor without and with different concentration of tomato juice. Tomato juice exerted an adverse effect on physical characteristics of sperm during refrigeration preservation. All the three sperm traits studied however revealed significant (p less thaN 0.01) positive interrelationships with correlations of 0.31 to 0.72.

Comparative efficacy of two extenders on post-thaw sperm characteristics of cryopreserved Black Bengal buck semen

The research work was conducted at the Artificial Insemination (AI) Center under the Department of Animal Breeding and Genetics, Bangladesh Agricultural University, Mymensingh to compare the efficacy between Egg Yolk Citrate (EYC) and Tris extenders for cryopreserved semen production in Black Bengal buck. The parameters of semen characteristics included percentage of individual motility (progressive), normal and live sperm and sperm concentration (billion/ml). Statistical analysis of fresh semen of individual buck effect was found to be significant (p&lt;0.01) on normal sperm percentage but insignificant on individual motility, sperm concentration and live sperm percentage. After dilution with EYC extender, individual bucks had significant effect (p&lt;0.05) on sperm motility and but insignificant on motility after cooling with and without glycerol. In the same way with Tris extender, it was insignificant (p&gt;0.05) on diluted semen motility and motility after cooling with and without glycerol. Motility and morphology of the sperm after equilibration and thawing showed insignificant difference among the bucks using EYC extender. On the other hand, variation in the motility after equilibration and thawing was found significant (p&lt;0.05) using Tris extender and insignificant on normal and live sperm percentages.&#x0D; Res. Agric., Livest. Fish.6(1): 119-125, April 2019

Fact findings about the frozen buck semen characters freezing with eyc and tris extender and productivity of Black Bengal does as the potential genetic resource in Bangladesh

The research work was conducted at the Artificial Insemination Center under the Department of Animal Breeding and Genetics, Bangladesh Agricultural University, Mymensingh to compare the efficacy between Egg Yolk Citrate (EYC) and Tris diluter for frozen semen production in Black Bengal buck. The parameters of semen characteristics included volume per ejaculate (ml), percentage of individual motility (progressive), normal and live sperm and sperm concentration (billion/ml). After dilution with EYC extender, It revealed from statistical analysis of frozen semen that individual bucks had significant effect (p&lt;0.05) on sperm motility and but insignificant on motility after cooling with and without glycerol. In the same way, with Tris diluter, it was insignificant (p&gt;0.05) on diluted semen motility and motility after cooling with and without glycerol. Motility and morphology of the sperm after equilibration and thawing showed insignificant difference among the bucks using EYC diluter. On the contrary, variation in the motility after equilibration and thawing was found significant (p&lt;0.05) using Tris diluter and insignificant on normal and live sperm percentages. After insemination with frozen buck semen, productivity or conception rate was found significantly (p&lt;0.01) higher (60.37%) in Tris than that of EYC diluter (43.75%). On the other hand, the productivity when compared among bucks within the diluter, the variation was not found significant (p&gt;0.05).The productivity found both in Tris and EYC diluter is almost similar to abundantly used Triladyl diluter (58.25). It is concluded that tris diluter might be used as the alternative to the Tryladil diluter though further study is to be needed for more confirmation.&#x0D; Res. Agric., Livest. Fish.5(3): 341-350, December 2018

Qualitative ultrastructural changes and morphometry of deccani sheep spermatozoa preserved with egg yolk citrate extender

The present investigation was aimed to study the sequential changes in the sperm cell deterioration dur-ing liquid storage of Deccani sheep breed semen from dilution to 48 h of storage along with its seminal characteris-tics and sperm morphometric measurement. Thus the two Deccani adult rams (aged 2 years), were selected (six ejaculates/each ram) and the collected semen was diluted with Egg yolk citrate extender (EYC) (final concentration - 400 million spermatozoa/0.2 ml semen).Seminal characteristics were assessed along with sperm morphologi-cal changes by Electron microscopy immediately after dilution, at 24 and 48 h of storage, respectively. Sperm morphometry was analysed by Image analysis.The percentage of Individual motility, Live spermatozoa, Acrosomal integrity and Hos-test reactive sperm decreased significantly (P&lt;0.05) from 80.41 to 49.16%, 82.75 to 51.25%, 94.16 to 83% and 76 to 48.58%, respectively during liquid storage of semen from initial dilution to 48 h of storage. The sperm head length (?m), Head width, sperm head area (?m2), sperm head perimeter (?m), mid piece length (?m), proximal mid piece width (?m), distal midpiece width (?m), volume of mid piece (?m3) and acrosomal cap length (?m) were 7.80, 4.33, 26.84, 20.63, 14.03, 0.74, 0.51, 4.54 and 5.24, respectively. Electron microscopic qualitative evaluation revealed that the main site of injury is the apical ridge of ram spermatozoa when stored at 5ºC. The electron density of the mitochondria reduced indicating concomittant depletion of ATP and loss of motility resulting in reduction of fertility.

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.