Journal articles on the topic 'Frozen thawed bull seman'
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Turk, G., M. Yuksel, M. Sonmez, S. Gur, S. Ozer Kaya, and E. Demirci. "Effects of semen sexing kits (HeiferplusTM and BullplusTM) supplemented to frozen-thawed bull semen on pregnancy rates, foetal sex ratios and selected reproductive parameters in cows." Veterinární Medicína 60, No. 6 (July 15, 2016): 309–13. http://dx.doi.org/10.17221/8245-vetmed.
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Ntemka, A., G. Tsousis, C. Brozos, E. Kiossis, CM Boscos, and IA Tsakmakidis. "Breed differences of bull frozen-thawed semen." Reproduction in Domestic Animals 51, no. 6 (September 25, 2016): 945–52. http://dx.doi.org/10.1111/rda.12769.
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Morató, Roser, Noelia Prieto-Martínez, Rodrigo Muiño, Carlos O. Hidalgo, Joan E. Rodríguez-Gil, Sergi Bonet, and Marc Yeste. "Aquaporin 11 is related to cryotolerance and fertilising ability of frozen–thawed bull spermatozoa." Reproduction, Fertility and Development 30, no. 8 (2018): 1099. http://dx.doi.org/10.1071/rd17340.
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Aquaporins (AQPs) are channel proteins involved in the transport of water and solutes across biological membranes. In the present study we identified and localised aquaporin 11 (AQP11) in bull spermatozoa and investigated the relationship between the relative AQP11 content, sperm cryotolerance and the fertilising ability of frozen–thawed semen. Bull ejaculates were classified into two groups of good and poor freezability and assessed through immunofluorescence and immunoblotting analyses before and after cryopreservation. AQP11 was localised throughout the entire tail and along the sperm head. These findings were confirmed through immunoblotting, which showed a specific band of approximately 50 kDa corresponding to AQP11. The relative amount of AQP11 was significantly (P < 0.05) higher in both fresh and frozen–thawed spermatozoa from bull ejaculates with good freezability compared with those with poorer freezability. In addition, in vitro oocyte penetration rates and non-return rates 56 days after AI were correlated with the relative AQP11 content in fresh spermatozoa. In conclusion, AQP11 is present in the head and tail of bull spermatozoa and its relative amount in fresh and frozen–thawed spermatozoa is related to the resilience of the spermatozoa to withstand cryopreservation and the fertilising ability of frozen–thawed spermatozoa. Further research is needed to elucidate the actual role of sperm AQP11 in bovine fertility.
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Siqueira, L. G. B., C. W. Palmer, and C. Lessard. "222 THE USE OF IN VITRO FERTILIZATION TO STUDY BOVINE IDIOPATHIC INFERTILITY." Reproduction, Fertility and Development 21, no. 1 (2009): 209. http://dx.doi.org/10.1071/rdv21n1ab222.
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A 4-year-old beef bull underwent a breeding soundness evaluation at the Western College of Veterinary Medicine (Veterinary Hospital, University of Saskatchewan); no apparent abnormalities were observed after conventional semen evaluations. However, the clinical history of this bull indicated that no pregnancies resulted from natural service of 52 cycling cows over a period of 2 years. Completed services were observed. The objective of this study was to use in vitro fertilization (IVF) technology to evaluate whether the sperm from this infertile bull could successfully fertilize oocytes in vitro. Fresh semen was collected with an electroejaculator and frozen in a computer-controlled rate freezer. Concentration and motility parameters were assessed by using computer-assisted semen analyses. Sperm morphology was evaluated on eosin-nigrosin-stained slides, and Coomassie blue staining was used to evaluate the presence of intact acrosomes. Within each evaluation technique, frozen–thawed semen from bulls (n = 2) with proven fertility was used as a positive control and samples of dead sperm (produced by repeated frozen–thawed cycles until reaching no sperm motility) were used as a negative control. Frozen semen from the infertile bull was used for IVF assay. Data were analyzed by ANOVA (sperm motility and the presence of acrosomes) or the chi-square test (cleavage and blastocyst rates), with a P-value of 0.05. Our infertile bull showed an average motile sperm percentage of 91.7 ± 2.2%, with 78.6 ± 3.5% progressive motility. After cryopreservation procedures, frozen–thawed semen had an acceptable general and progressive percentage of motility of 57.8 ± 6.7% and 43.9 ± 9.2%, respectively. Sperm stained with Coomassie blue showed a greater proportion of intact acrosomes in the fresh semen (63.6 ± 4.3% v. 40.4 ± 3.7%; P < 0.05); however, frozen–thawed semen from both the fertile bull and the control were similar (40.4 ± 3.7b% v. 45.5 ± 2.2b%, P < 0.05). In vitro fertilization results revealed a low cleavage rate at 48 h postfertilization (19.8 ± 6.3%) and blastocyst rate (2.4 ± 2.8%) when using frozen–thawed semen from the infertile bull, compared with the control (56.7 ± 8.2% and 26.3 ± 4.5%, respectively; P < 0.001). Moreover, cleavage and blastocyst rates obtained by using the negative control (21.1 ± 3.2% and 1.1 ± 1.9%, respectively) were similar to those of the infertile bull (P > 0.10). It was noted that ova fertilized with either frozen–thawed semen from the infertile bull or the negative control did not produce blastocysts before Days 8 and 9 of embryo culture, which is a characteristic of parthenogenesis. The results from this IVF study suggest that in this bull, there was a missing or defective factor blocking one of the steps in the fertilization process. Further investigations to identify this factor will increase our knowledge of male fertility, and could lead to new methods of evaluating and regulating fertilizing ability.
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Mphaphathi, M. L., M. M. Seshoka, T. R. Netshirovha, Z. C. Raphalalani, T. C. Chokoe, M. Nkadimeng, N. L. Kanuya, J. P. C. Greyling, and T. L. Nedambale. "37 DOUBLE FREEZING AND THAWING OF NGUNI BULL SEMEN." Reproduction, Fertility and Development 28, no. 2 (2016): 148. http://dx.doi.org/10.1071/rdv28n2ab37.
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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 < 0.05). The sperm curvilinear velocity and average path velocity was reduced following first frozen-thawed (P < 0.05) but remained constant and stable between the treatment groups (P > 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.
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Arat, S., S. Pabuccuoglu, H. Sagirkaya, K. Demir, R. Arici, B. Ustuner, S. Alcay, et al. "22 SEMEN AND REPRODUCTIVE PROFILES OF CLONED ANATOLIAN GREY CATTLE." Reproduction, Fertility and Development 27, no. 1 (2015): 103. http://dx.doi.org/10.1071/rdv27n1ab22.
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Anatolian grey cattle (endangered native Anatolian cattle) as 1 male (clone 1) and 4 females (clones 2–5) were produced from cells of 1 male and 1 female cattle by somatic cell nuclear transfer (SCNT) in a previous study. In this study, we examined the reproductive potential of these cloned animals, which are now 4 and 5 years old. The parameters evaluated by phase contrast microscopy for motility, TUNEL for DNA fragmentation, eosin staining for viability, Hoechst 33258 staining and hypo-osmotic swelling test (HOST) for membrane integrity, and fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA) for acrosome integrity of frozen-thawed spermatozoa, as well as birth and survival of calves following insemination with frozen-thawed semen of cloned and nuclear donor bull and normal bull. Six ejaculates and 3 samples per ejaculate from each bull were tested, and the Mann-Whitney U test was used to analyse the data. The spermatological parameters of cloned bull semen – volume, concentration, and motility of fresh – were within accepted limits for artificial insemination (4.60 ± 0.47 mL, 1.55 ± 0.21 × 109 spermatozoa mL–1, 80.00 ± 1.07%, respectively). Frozen-thawed sperm motility and viability rate were higher in the cloned bull (56.6%, 56.7%) than in its nuclear donor (47%, 43%; P < 0.05). Intact membrane and DNA fragmentation rate of cloned bull and its nuclear donor bull sperm were similar (P > 0.05) but the intact acrosome rate of cloned bull was higher than that of its nuclear donor (P < 0.05). Low rates in frozen-thawed sperm of nuclear donor can be related to storage time of sperm which were frozen 5 years before. One (clone 4) of the cloned grey heifers was artificially inseminated with frozen semen from nuclear donor bull and the other (clone 5) was naturally mated with a Holstein bull. Two healthy calves were delivered naturally. When same cloned cows (clones 4–5) and 2 other cloned heifers (clones 2–3) were artificially inseminated with frozen semen of the cloned grey bull, clones 2 and 4 gave birth to 2 healthy female calves. One cloned cow (clone 3) aborted in the third month of gestation and other one (clone 5) is currently 8 months pregnant. Two calves of clone 4 and 5 are 17 months old and 2 other calves of clone 2 and 4 are now 6 and 1 months old. Except for clone 3, our results show that cloned Anatolian grey bull and cows produced from frozen cells in gene bank have normal fertility.
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Malcolm, V., M. Marfil, M. Calvi, F. Rigali, M. Pugliese, J. Gutierrez, M. Panarace, and M. Medina. "365 COMPARISON OF IN VITRO FERTILIZING CAPACITY OF FROZEN - THAWED SEX-SORTED AND SEX-SORTED FROZEN - THAWED BULL SPERMATOZOA." Reproduction, Fertility and Development 19, no. 1 (2007): 298. http://dx.doi.org/10.1071/rdv19n1ab365.
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Sperm sexing has become a world-wide technology, now available in many countries. The method has been incorporated into many reproductive technologies such as embryo production (Zhang et al. 2003 Theriogenology 60, 1657–1663), but sex-sorting is limited when bulls are located far from sorters or when only frozen semen is available. Previous studies on sexing frozen–thawed spermatozoa have been done in rams, which resulted in retention of the spermatozoan functional capacities (Hollinshead et al. 2004 Reproduction 127, 557–568). In vitro characteristics were assessed in bulls after sexing of thawed sperm (Hollinshead et al. 2004 Theriogenology 62, 958–968); however, the fertilizing capacity of frozen–thawed sex-sorted (FTSS) spermatozoa was not tested. The aim of the present study was to compare cleavage and embryo development rate among frozen–thawed (FT), sex-sorted frozen–thawed (SSFT), and FTSS bull spermatozoa. For FT, sperm were diluted to a final concentration of 60 × 106 sperm/mL, packaged in 0.5-mL straws, and frozen. In SSFT, spermatozoa were sex-sorted by flow cytometry following Schenk protocols (1999 Theriogenology 52, 1375–1391). Three × 106 spermatozoa were packaged into 0.25-mL straws and frozen. The final treatment (FTSS) consisted of thawing 6 to 10 frozen straws of 4 different bulls containing an average of 25 × 106 spermatozoa and centrifuging at 600g for 15 min at 21°C to extract cryodiluent. Spermatozoa were diluted and stained with Hoechst 33342 (stain concentration of 112.5 µM, the same used for SSFT treatment) following Schenk sexed-semen protocols (1999), sex-sorted by a flow cytometer, and collected in Tris-base extender containing 20% egg yolk. For each ejaculate frozen–thawed, SSFT and FTSS spermatozoa were prepared for oocyte in vitro fertilization. Also, semen from a bull routinely used as a control in the laboratory was added for a better comparison of results. Oocytes from a slaughterhouse were processed following standard in vitro fertilization procedures (Ferré 2002 Theriogenology 57, 664) 4 times for each bull, and comparison was made between treatments. Results were analyzed by ANOVA. No significant differences were observed among bulls (data not shown) (P > 0.05). Although embryo development rate was statistically different between sexed and non-sexed groups (P < 0.05), results showed that frozen–thawed bull spermatozoa can be sex-sorted and used for in vitro fertilization with comparable developmental rates comparable to those when frozen sexed semen is used (Table 1). This opens a new commercial window for cases where pre-selected sexed embryos from bulls that are not in AI centers are desired, also giving an opportunity for dead bulls. Nevertheless, since a large number of straws are necessary, further studies must be carried out to make this procedure more efficient and economically profitable. Table 1. Results of cleavage and embryo development rates between spermatozoa treatments
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Underwood, S. L., R. Bathgate, D. C. Pereira, A. Castro, P. C. Thomson, W. M. C. Maxwell, and G. Evans. "Embryo production after in vitro fertilization with frozen-thawed, sex-sorted, re-frozen-thawed bull sperm." Theriogenology 73, no. 1 (January 2010): 97–102. http://dx.doi.org/10.1016/j.theriogenology.2009.08.005.
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Öztürk, Caner, Şükrü Güngör, Mehmet Bozkurt Ataman, Mustafa Numan Bucak, Nuri Başpinar, Pınar Ili, and Muhammed Enes Inanç. "Effects of arginine and trehalose on post-thawed bovine sperm quality." Acta Veterinaria Hungarica 65, no. 3 (September 2017): 429–39. http://dx.doi.org/10.1556/004.2017.040.
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The present study was conducted to examine the protective role of arginine and trehalose on post-thaw bull sperm and oxidative stress parameters. Five ejaculates for each bull were used in the study. Each ejaculate, split into three equal aliquots and diluted at 37 °C with base extenders containing 2 mM arginine, 25 mM trehalose and no antioxidant (control) was cooled to 5 °C and then frozen. Frozen straws were thawed in a water bath for evaluation. Supplementation of the semen extender with arginine decreased the percentages of post-thawed subjective motility (29 ± 8.21%), CASA motility (12.2 ± 5.69%) and progressive motility (3.52 ± 2.13%), compared with the controls (43 ± 2.73%, 55.4 ± 6.78% and 33.48 ± 4.14%, respectively, P < 0.05). Supplementation of the semen extender with trehalose produced a higher mitochondrial activity and sperm viability (36.3 ± 3.99% and 44.1 ± 2.18%) compared with the control (13 ± 8.15 and 31.7 ± 3.94%, respectively, P < 0.05). It was established that trehalose (95.1%) and arginine (92.8%) protect DNA integrity compared to the control (90.4%) (P < 0.05). Trehalose supplementation in semen extenders provided great benefit in terms of viability, mitochondrial activity, and intact sperm DNA on frozen-thawed bull sperm.
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Kaka, Asmatullah, Wahid Haron, Rosnina Yusoff, Nurhusien Yimer, A. M. Khumran, Kazhal Sarsaifi, Atique Ahmed Behan, Ubedullah Kaka, Akeel Ahmed Memon, and Mahdi Ebrahimi. "Effect of docosahexanoic acid on quality of frozen–thawed bull semen in BioXcell extender." Reproduction, Fertility and Development 29, no. 3 (2017): 490. http://dx.doi.org/10.1071/rd15089.
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This study was conducted to investigate the effect of docosahexanoic acid (DHA) supplementation in BioXcell extender on the quality of frozen–thawed bull semen. Twenty-four ejaculates were collected from three bulls (eight from each bull). Ejaculates with motility ≥70% and normal morphology ≥80% were extended into BioXcell extender to which 0 (control), 3, 5, 10 or 15 ng mL–1 DHA was added. The supplemented semen samples were incubated at 37°C for 15 min for DHA uptake by spermatozoa. Later, samples were cooled for 2 h at 5°C and packaged into 0.25-mL straws, frozen in liquid nitrogen for 24 h and subsequently thawed for evaluation. Results are presented as percentages ± s.e.m. Supplementation with DHA at 3 ng mL–1 significantly improved sperm functional parameters including sperm motility, normal morphology, viability, acrosome integrity and membrane integrity when compared with other supplemented groups and the control. Lipid peroxidation increased as the incorporation of DHA supplementation increased. In conclusion, 3 ng mL–1 concentration of DHA resulted in superior quality of frozen–thawed bull spermatozoa and is suggested as the optimum level of DHA to be added into BioXcell extender.
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Yoon, Jae-Wook, Seung-Eun Lee, Won-Jae Kim, Dae-Cheol Kim, Cheol-Ho Hyun, Shin-Ji Lee, Hyo-Jin Park, et al. "Evaluation of Semen Quality of Jeju Black Cattle (JBC) to Select Bulls Optimal for Breeding and Establish Freezing Conditions Suitable for JBC Sperm." Animals 12, no. 5 (February 22, 2022): 535. http://dx.doi.org/10.3390/ani12050535.
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To optimize the reproduction of Jeju black cattle (JBC), freezing conditions for sperm were established and sperm motility, vitality, morphology, and fertility were evaluated to select the optimal bull for breeding. Semen samples from five JBC bulls were individually mixed with freezing medium at a final concentration of 1 × 108 sperm/mL and frozen in liquid nitrogen vapor at a height of 3 or 7 cm (referred to as 3 cm sperm and 7 cm sperm, respectively). When the freezing conditions were compared, the motility of 7 cm sperm was significantly higher than that of 3 cm sperm for the JBC-A bull. The motility, curvilinear velocity, straight-line velocity, and average path velocity of fresh and frozen–thawed sperm were the highest for the JBC-A bull. The vitalities of fresh and frozen–thawed sperm were the highest for the JBC-A/E and JBC-A bulls, respectively. The percentage of normal cells in fresh sperm was the highest for the JBC-D bull. The rates of the normal formation of two pronuclei and total sperm penetration were the highest in zygotes fertilized with sperm from the JBC-A bull. The sperm from the JBC-A bull had superior qualities and are thus the most appropriate choice for the preservation and reproduction of these endangered cattle.
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McCue, P., J. Kelly, S. Ashworth, D. Kleemann, and S. Walker. "249 EFFECT OF REFREEZING BULL SEMEN ON IVF SUCCESS RATE." Reproduction, Fertility and Development 17, no. 2 (2005): 275. http://dx.doi.org/10.1071/rdv17n2ab249.
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Cryopreserved semen from valuable sires may be available in limited quantities in some situations. A large percentage of the spermatozoa in a thawed straw is potentially wasted since a relatively small number of spermatozoa are required for most assisted reproduction techniques. The goal of this study was to determine the effect of dilution and refreezing of bull semen on fertilization and blastocyst development rates following in vitro fertilization. The hypothesis was that frozen bull semen that was thawed, diluted, and refrozen could be used successfully for IVF. Oocytes were harvested from cow ovaries collected from a local abattoir and matured in vitro for 24 hours. Ova were subsequently assigned to one of four in vitro fertilization treatment groups. Group 1 ova (n = 158) were fertilized with bull semen frozen at a concentration of 20 × 106 spermatozoa per 0.25 mL straw. Group 2 ova (n = 157) were fertilized with semen frozen at an initial concentration of 2 × 106 spermatozoa. Group 3 ova (n = 157) were fertilized with semen that had been thawed and refrozen at a concentration of 20 × 106 spermatozoa. Group 4 ova (n = 150) were fertilized with semen that had initially been frozen at a concentration of 20 × 106 spermatozoa and then thawed, diluted to a concentration of 2 × 106 spermatozoa, and refrozen. IVF was performed in a medium volume of 100 μL using 1 × 106 spermatozoa/mL. Cleavage and blastocyst rates were determined 2 days and 7 days, respectively after IVF. Cleavage rates following IVF was highest with semen frozen at 20 × 106 spermatozoa (89.9%), intermediate with semen frozen at 2 × 106 spermatozoa or refrozen at 20 × 106 spermatozoa (71.3% and 73.9%, respectively), and lowest with semen refrozen at 2 × 106 spermatozoa (38.7%) (P < 0.05). Blastocyst development rate was not significantly different (P > 0.05) among treatment groups. This study confirmed the hypothesis that refrozen bovine semen can be used successfully for in vitro fertilization. Although the overall IVF efficiency was lower using diluted refrozen semen, multiple IVF procedures could theoretically be performed over time from one initial straw. Consequently, if a limited amount of frozen semen is available, thawing of a single straw followed by dilution, re-allocation into multiple straws, and refreezing should be considered to facilitate the more efficient use of semen in future assisted reproduction endeavors. This study was supported by the PEG Program, Colorado State University.
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Jankovičová, Jana, Michal Simon, Jana Antalíková, and Ľubica Horovská. "Acrosomal and viability status of bovine spermatozoa evaluated by two staining methods." Acta Veterinaria Hungarica 56, no. 1 (March 1, 2008): 133–38. http://dx.doi.org/10.1556/avet.56.2008.1.14.
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Artificial insemination with frozen-thawed spermatozoa is commonly used in cattle breeding. A simple and fast procedure is needed for routine evaluation of the acrosomal status of frozen-thawed bovine sperm. Therefore, the purpose of this study was to test two staining procedures used to determine the viability and integrity of acrosome of frozen-thawed bovine spermatozoa. Double staining and Hoechst/FITC- Pisum sativum agglutinin (FITC-PSA) labelling were tested for evaluating the viability and acrosome reaction induced by calcium ionophore of bull spermatozoa. In our experiments no significant differences were detected in the frequency of acrosome-reacted sperm either by double staining (37.98%) or by FITC-PSA labelling (39.33%). The viability of sperm stained by the double staining method was 67.17%, and a higher portion of viable sperm (82.67%) was observed by staining with the Hoechst procedure (P < 0.01). On the basis of the results obtained it is concluded that both methods can be used for detecting the acrosome reaction of frozen-thawed bovine spermatozoa.
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Baiee, Falah, Abd Wahid Haron, Murtadha A. AL-mudhafr, Innocent Damudu Peter, and Nurhusien Yimer. "Can Panax Ginseng Aqueous Extract Improve Chilled and Cryopreserved Bull Spermatozoa?" Agricultural Science 2, no. 2 (July 14, 2020): p15. http://dx.doi.org/10.30560/as.v2n2p15.
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This study was to evaluate the influence of Panax ginseng aqueous extract on chilled and frozen-thawed bull sperm quality. Samples of semen were acquired from four bulls through the use of an electro-ejaculator. Extension of the semen was done with tris-egg yolk diluent which was augmented with 0.0, 0.25, 0.5, 1.0, 2.5, 5.0, and 7.5 mg/mL Panax ginseng aqueous extract. Diluted chilled portions of the semen were chilled for 6 days at 5 ̊C whereas the frozen semen was cryopreserved in liquid nitrogen. Results revealed that in chilled and frozen-thawed semen, the control group, T1 and T2 recorded higher percentages in terms of sperm motility and viability in all three groups evaluated compared to others, while the high dose of Panax ginseng aqueous extract in T6 and T5 recorded the lowest percentage. Moreover, the values of sperm morphology for chilled and frozen-thawed semen were not significant among the groups. The results of chromatin stability of the present study showed that T2 and control were higher than for other groups. In conclusion, the low dosage groups (T1, T2 and T3) which were received (0.25 mg/mL, 0.5 mg/mL and 1 mg/mL, respectively) from Panax ginseng aqueous extract were not significant as compared with the control group while high-dosage groups (T4, T5 and T6) which were received (2.5 mg/mL, 5 mg/mL and 7.5 mg/mL, respectively) from Panax ginseng aqueous extract were highly decreased spermatozoa characteristics.
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Underwood, S. L., R. Bathgate, M. Ebsworth, W. M. C. Maxwell, and G. Evans. "Pregnancy loss in heifers after artificial insemination with frozen-thawed, sex-sorted, re-frozen-thawed dairy bull sperm." Animal Reproduction Science 118, no. 1 (March 2010): 7–12. http://dx.doi.org/10.1016/j.anireprosci.2009.06.004.
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Underwood, S. L., R. Bathgate, W. M. C. Maxwell, and G. Evans. "Birth of offspring after artificial insemination of heifers with frozen-thawed, sex-sorted, re-frozen-thawed bull sperm." Animal Reproduction Science 118, no. 2-4 (April 2010): 171–75. http://dx.doi.org/10.1016/j.anireprosci.2009.08.007.
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KUMAR, N., A. K. SINGH, RANJNA S. CHEEMA, A. KUMAR, H. KAUR, and P. S. BRAR. "Impact of dietary feeding of vitamin E in buffalo bulls on fresh and frozen-thawed semen characteristics and antioxidant status." Indian Journal of Animal Sciences 88, no. 6 (June 22, 2018): 677–83. http://dx.doi.org/10.56093/ijans.v88i6.80883.
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Vitamin E is the main chain-breaking, naturally occurring free radical scavenger that has significant biological implications on sperm. However, its role as an antioxidant on semen quality of buffalo bulls is still obscure. The present study was undertaken to investigate the effect of dietary feeding of vitamin E on fresh- and frozen-thawed semen characteristics, and antioxidant status in buffalo bull. Six apparently healthy breeding Murrah buffalo bulls were randomly selected at University bull farm for the present study. The bulls were divided into two groups, viz. control group (n = 3) and feeding group (n = 3). The bulls of feeding group were fed vitamin E @ 4000 IU/bull/day for 60 days. Accordingly, 120 ejaculates (one ejaculate/bull/session) were collected from bulls of control and feeding groups during pre-feeding, feeding and post-feeding phase of vitamin E and analyzed for semen characteristics and oxidative stress. Most beneficial effects of dietary feeding of vitamin E were observed during post-feeding phase. The percentages of total and progressive motility, viability, plasma membrane integrity, malondialdehyde (MDA) and glutathione peroxidase (GPX) in bulls fed with vitamin E were significantly higher than in their control counterparts during post-feeding phase of fresh and frozen-thawed semen. The levels of same parameters were also significantly higher as compared to that during feeding stage in fresh- and frozen-thawed semen of feeding group. It is therefore concluded that feeding vitamin E to buffalo bulls protected sperm membrane against oxidative damage and improved the fertilizing potential of spermatozoa.
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Ahmad, Mushtaq, Nasim Ahmad, Amjad Riaz, and Muhammad Anzar. "Sperm survival kinetics in different types of bull semen: progressive motility, plasma membrane integrity, acrosomal status and reactive oxygen species generation." Reproduction, Fertility and Development 27, no. 5 (2015): 784. http://dx.doi.org/10.1071/rd13400.
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This study was designed to compare the kinetics of sperm survival in different types of bull semen. Fresh ejaculates from four bulls were pooled, diluted in Tris-citric acid-egg yolk-glycerol extender, cooled to 4°C, frozen in LN2 and thawed at 37°C. Fresh, diluted, cooled and frozen–thawed semen were incubated at 37°C, and evaluated at 0, 2, 4, 6, 12 and 24 h after the beginning of incubation. In Experiment 1, progressive sperm motility, normal acrosomes and plasma membrane integrity and asymmetry were determined. In Experiment 2, generation of superoxide anion (O2•) along with plasma membrane permeability and generation of hydrogen peroxide (H2O2) along with plasma membrane integrity were assessed. In Experiment 1, frozen–thawed semen had shorter survival times for progressive sperm motility, and spermatozoa with intact plasma membranes and acrosomes (IPM-IACR) as compared with other types of semen (P < 0.05). Fresh spermatozoa underwent a necrotic pathway, diluted and cooled spermatozoa underwent an apoptosis-like pathway and frozen–thawed spermatozoa underwent both necrotic and apoptosis-like pathways. In Experiment 2, spermatozoa in all four types of semen exhibited O2•– generation and increased plasma membrane permeability, and became necrotic without H2O2 generation during incubation (P < 0.05). In conclusion, frozen–thawed semen had shorter sperm longevity, which has important implications relating to the timing of artificial insemination. Different types of semen followed different death pathways. During incubation, spermatozoa in all types of semen generated O2•–, which increased the permeability and compromised the integrity of the plasma membrane.
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Khumran, A. M., N. Yimer, Y. Rosnina, H. Wahid, M. O. Ariff, H. Homayoun, K. Asmatullah, and T. K. Bello. "Butylated hydroxytoluene protects bull sperm surface protein-P25b in different extenders following cryopreservation." April-2020 13, no. 4 (2020): 649–54. http://dx.doi.org/10.14202/vetworld.2020.649-654.
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Aim: The aim of this study was to investigate the effects of different concentration of butylated hydroxytoluene (BHT) on sperm membrane surface protein "P25b" from cryopreserved bull semen in either lecithin based Bioxcell® (BX) or two egg-yolk based extenders, tris-egg yolk (TEY), and citrate-egg yolk (CEY). Materials and Methods: Forty-five semen samples, 15 each were extended with either BX, TEY, or CEY extender which contained different concentrations (0.0 - control, 0.5, 1.0, 1.5, 2.0, and 3.0 mM/mL) of BHT. The extended semen samples were frozen at a concentration of 20×106/mL in 0.25 mL straws and stored in liquid nitrogen for 2 weeks. The frozen samples were thereafter thawed, proteins extracted and analyzed for quantities of protein P25b through direct sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel densitometry. Peptides were confirmed by Western blotting (WB). Results: Results showed that supplementation of BHT improved (p<0.05) quantity of protein P25b at concentrations of 0.5 mM/mL for BX and at 1.0 mM/mL for TEY and CE when compared with the controls and other treatments. Conclusion: BHT supplementation at 0.5 in BX and 1.0 mM/mL in TEY and CEY has protected bull sperm fertility marker protein P25b in frozen-thawed bull sperm.
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Hayama, K., M. Takeuchi, A. Ideta, M. Urakawa, M. Sasatani, Y. Nagamune, and Y. Aoyagi. "294 EFFECT OF SPERMATOZOA MOTILITY ON TRANSFERABLE EMBRYO RATE OF SUPEROVULATED JAPANESE BLACK CATTLE." Reproduction, Fertility and Development 21, no. 1 (2009): 244. http://dx.doi.org/10.1071/rdv21n1ab294.
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Sperm motility is known to affect fertilization; however, little is known about the relationship between frozen–thawed sperm motility and in vivo fertilization following superovulatory treatment. The objective of this study was to evaluate a sperm function test as potential predictors of embryo production following superovulatory treatment in cattle. Two to five batches of semen (Japanese black bull, n = 4, A to D) were diluted with egg york-citrate-glycerol in 0.5 mL plastic straws, and they were stored in liquid nitrogen until analyzed. Frozen–thawed spermatozoa were evaluated for motility {motile sperm concentration (MSC, million mL–1), progressive MSC (PMSC, million mL–1) and velocity (μm s–1)} using a sperm quality analyzer for bulls (SQA-Vb, Medical Electronic Systems, Caesarea, Israel). Each sample of 20 μL aspirated into the disposable capillary, was inserted into SQA-Vb. Measurements were displayed within 75 s. Intra-assay CVs of MSC, PMSC, and velocity were 14.2, 7.3 and 7.5%, respectively. Inter-assey CVs of them were 13.5, 3.9 and 4.3% respectively. Superstimulated donors (Japanese black cows, n = 161) were artificially inseminated with one dose of frozen–thawed semen (bull A = 74, B = 46, C = 21 and D = 20). The proportion of transferable embryo (IETS grade 1 to 3) was examined on day 7 (day 0 = estrus). Data were analyzed using ANOVA followed by Scheffe multiple comparison test, and Fisher’s z-transformation. MSC, PMSC and velocity values differed significantly among each bull. The values of bull A were much lower than those of the other bulls. The proportion of transferable embryos produced by bull A was significantly lower than that of other bulls (P < 0.05, Table 1). Correlations showed significant association between MSC and proportion of transferable embryos (r = 0.99, P < 0.01). We conclude that bovine sperm motility using a SQA-Vb is a useful predictor of embryo production following superovulatory treatment. Table 1.Relationship between sperm motility and proportion of transferable embryo
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Monterroso, V. H., K. C. Drury, A. D. Ealy, J. L. Edwards, and P. J. Hansen. "Effect of heat shock on function of frozen/thawed bull spermatozoa." Theriogenology 44, no. 7 (November 1995): 947–61. http://dx.doi.org/10.1016/0093-691x(95)00282-d.
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Yasri, Rakhmi, Ristika Handarini, and Muhammad Imron. "THE QUALITY OF EMBRYOS RESULTED FROM IN VITRO FERTILIZATION BY USING FROZEN SEMEN THAWED IN DIFFERENT TEMPERATURES." Jurnal Peternakan Nusantara 3, no. 1 (October 13, 2017): 23. http://dx.doi.org/10.30997/jpnu.v3i1.853.
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In vitro fertilization technology in cows is an effort done to utilize ovary waste from cows slughtered in abbatoir. This study was aimed at assessing the qualiy of embryos resulted from in vitro fertilization by using frozen semen thawed in different temperatures. In order to get qualty semen, standardized thawing method is required. It was expected from this study that an optimum thawing temperature for frozen semen was determined to obtain quality transferable embryos. Three treatments consisting of thawing with water 37°C for 30 second (T1), thawing with water 25°C for 30 second (T2), and thawing with water 10°C for 30 second (T3). Data were subjected to an an anlysis of variance (Anova) and a Duncan test. Results showed that oocytes fertilized with frozen semen thawed at 37°C and 10°C had higher fertilization rate and excellent-grade embryos (P<0.05) than did the ones fertilized with frozen semen thawed at 25°C. However, no different effect of thawing temperatures was found on transferable and degenerated embryos (P>0.05). It was concluded that embryos fertilized with Brahman frozen semen in thawed at 37°C had the highest number of embryos (49.66±2.88) and excellent-grade embryos (22.00±4.35). Key words: Embryo quality, In vitro fertilization, frozen semen thawing, Brahman bull.
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Goldman, E. E., J. E. Ellington, P. B. Farrell, and R. H. Foote. "Use of fresh and frozen-thawed bivine oviduct cells for acrosome reacting fresh and frozen-thawed bull sperm in vitro." Theriogenology 35, no. 1 (January 1991): 204. http://dx.doi.org/10.1016/0093-691x(91)90180-l.
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Murphy, Craig, Shauna A. Holden, Edel M. Murphy, Andrew R. Cromie, Patrick Lonergan, and Sean Fair. "The impact of storage temperature and sperm number on the fertility of liquid-stored bull semen." Reproduction, Fertility and Development 28, no. 9 (2016): 1349. http://dx.doi.org/10.1071/rd14369.
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In Ireland, liquid bull semen is stored at unregulated ambient temperatures, typically at 5 × 106 spermatozoa per dose, and inseminated within 2.5 days of collection. In Experiment 1, the effect of storage temperature (5, 15, 22, 32°C and fluctuations (Flux) between these temperatures) on progressive motility, viability, acrosomal status, DNA fragmentation and osmotic resistance was assessed. In Experiment 2, the field fertility of liquid semen at 5, 4 and 3 × 106 spermatozoa per dose, up to Day 2 after collection, was assessed in comparison to frozen–thawed semen at 20 × 106 spermatozoa per dose (n = 35 328 inseminations). In Experiment 1, storage at 15°C resulted in the highest progressive motility (P < 0.01). The osmotic resistance of spermatozoa declined with duration of storage; however, after Day 3 this decline was reduced in the 5°C and Flux 15°C treatments (P < 0.01). In Experiment 2, the non-return rate of liquid semen stored at 4 and 3 × 106 spermatozoa per dose on Day 2 of storage was reduced in comparison to frozen–thawed semen (P < 0.01). In conclusion, liquid semen is versatile between storage temperatures of 5 and 22°C, but demonstrates reduced fertility on Day 2 of storage at lower sperm numbers in comparison to frozen–thawed semen.
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Nedambale, T. L., J. Xu, S. A. Chaubal, X. C. Tian, X. Yang, and F. Du. "307 ADDITION OF HIGH CONCENTRATIONS OF HEPARIN DURING IN VITRO FERTILIZATION IMPROVES PERCENTAGES OF CLEAVAGE AND BLASTOCYST FORMATION WHEN BOVINE OOCYTES ARE INSEMINATED IN VITRO WITH SEX-SORTED SPERM." Reproduction, Fertility and Development 19, no. 1 (2007): 269. http://dx.doi.org/10.1071/rdv19n1ab307.
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The developmental potential of oocytes fertilized in vitro by sexed–sorted frozen–thawed sperm remains low. The aim of this study was to find an optimal concentration of heparin during IVF using frozen–thawed sex-sorted sperm to improve the percentages of fertilization and blastocyst formation. A total of 1708 matured bovine oocytes were randomly allocated among different concentrations of heparin (0, 2.5, 5, 10, 20, and 40 µg mL−1) in Bracket-Olifant medium and co-incubated with non-sexed or sex-sorted sperm for 6 h (Day 0). The sperm (sorted or not) used for IVF was from one bull of proven fertility. Presumptive zygotes then were cultured in CR1aa + 6 mg mL−1 of BSA in 5% O2, 5% CO2, and 90% N2 at 39°C until Day 8. Percentages of cleavage were recorded on Day 2. Data (3 replicates) were analyzed by ANOVA. The results (Table 1) demonstrated that addition of different concentrations of heparin in IVF medium for non-sexed sperm groups did not alter cleavage and blastocyst formation. However, the lowest percentages of cleavage were observed with 0, 2.5, and 5 µg mL−1 of heparin in sexed sperm groups, demonstrating that high concentrations of heparin (20 and 40 µg mL−1) positively affected the percentages of cleavage and blastocyst formation. A greater proportion of zygotes in 20 and 40 µg mL−1 sexed sperm groups developed into grade 1 blastocysts on Day 7, and subsequently formed more blastocysts on Day 8 compared with 0, 2.5, and 5 µg mL−1 of heparin in sexed sperm groups. In conclusion, addition of heparin was not necessary when frozen–thawed non-sexed sperm were used for IVF with this particlar bull. However, addition of higher concentrations of heparin during IVF improved fertilization and blastocyst formation in vitro (40 µg mL−1 of heparin could be used as an alternative concentration for frozen–thawed sex-sorted sperm with this particular bull). The incidence of polyspermy as well as polyploidy in blastocysts is currently being studied. Table 1.Effect of heparin concentrations during IVF on cleavage and blastocyst formation
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Nedambale, T. L., M. L. Mphaphathi, P. H. Munyai, M. Tshabalala, P. Malusi, and A. Dinnyes. "107 MOTILITY PARAMETER PATTERNS OF NGUNI BULLS: EFFECT OF VARIOUS GYCEROL CONCENTRATIONS FOLLOWING CRYOPRESERVATION." Reproduction, Fertility and Development 22, no. 1 (2010): 212. http://dx.doi.org/10.1071/rdv22n1ab107.
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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 < 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).
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Kątska-Książkiewicz, L., B. Ryńska, M. Bochenek, J. Opiela, and J. Jurkiewicz. "In vitro production of bovine embryos using flow-cytometrically sexed sperm." Archives Animal Breeding 49, no. 2 (October 10, 2006): 133–40. http://dx.doi.org/10.5194/aab-49-133-2006.
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Abstract. The investigation aimed to compare the effect of fresh and frozen-thawed X and Y fractions of flow-cytometrically sorted bovine spermatozoa on in vitro fertilization of bovine in vitro matured oocytes and subsequent blastocyst development. Sperm cells sorted in MoFloSX cytometer were used either for IVF or frozen and stored in liquid nitrogen. Immature oocytes recovered from ovaries of slaughtered animals and matured in vitro in TCM-199 containing 20% estrus cow serum and additional granulosa cells were fertilized in vitro with fresh or frozen-thawed fractions of sorted sperm. Simultaneously, control, fresh or frozen/thawed sperm was used for IVF. A total number of 2712 IVM oocytes were fertilized with sorted and control sperm of 6 bulls. Embryo cleavage rates were significantly affected by bull (P<0.0001), sperm sexing (P<0.0001) and sperm freezing (P<0.01). Blastocysts development was affected by sperm freezing (P<0.04) and sperm sexing (P<0.01). The significant differences were shown between unsorted and sorted sperm, however no differences in embryo cleavage rates and blastocysts rates were observed between X- and Y-sperm fractions, both fresh and frozen/ thawed. There were significant differences in cleavage rates among fresh, control sperm (52.7%), X fraction (26.8%) and Y fraction (24.7%). Similar differences in cleavage rates were shown for frozen/thawed control sperm (52.8%), X fraction (33.9%) and Y fraction (26.2%). The female blastocysts were frozen for further transfer, while the hatched male blastocysts were analysed by PCR revealing 76.2% accuracy. The results suggest that there were significant differences in cleavage rates and blastocyst rates due to sperm sorting in comparison to unsorted sperm and no differences between effectiveness of X and Y fractions of spermatozoa.
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Petersen, M. M., G. B. Boe-Hansen, A. Birck, B. Avery, M. Jensen, and I. B. Bøgh. "309 DIFFERENCES IN PRONUCLEUS FORMATION RATES BETWEEN BULLS IN RESPONSE TO GAMMA IRRADIATION OF FROZEN - THAWED SEMEN." Reproduction, Fertility and Development 19, no. 1 (2007): 270. http://dx.doi.org/10.1071/rdv19n1ab309.
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Gamma irradiation induces DNA damage to mature bovine spermatozoa but does not affect motility, membrane integrity, or function (Fatehi et al. 2006 J. Androl. 27, 176–188), making it a useful model to evaluate the effect of fertilization with DNA-damaged spermatozoa. The objective of the present study was to analyze the fertilizing capacity of frozen–thawed gamma-irradiated sperm from 2 bulls: a bull with a high sperm quality and fertilization rate (bull A), and a bull with a low sperm quality and fertilization rate (bull B). To ensure that DNA damage was induced, frozen–thawed semen was exposed to a high dose of irradiation (90 Gy). Cumulus–oocyte complexes were obtained from abattoir ovaries and were in vitro-matured (IVM) using standard procedures (23 h in DMEM with 5% serum and eCG/hCG). For each treatment group, 3 to 5 straws of semen from the same ejaculate were used. After thawing, semen from the 2 bulls was either irradiated or held at room temperature before 1:1 dilution in sperm-TALP. During irradiation, semen was kept in the straws. The irradiated and nonirradiated sperm were used for IVF (23 h in IVF-TALP) within 1 h of thawing. IVM and IVF were carried out at 38.5�C in 5% CO2 in air. After IVF, presumptive zygotes were whole mount-fixed and 24 h later were stained with 1% aceto-orcein followed by determination of fertilization status. Fertilization was considered normal if 2 pronuclei (PN) were present. A total of 4 replicates were performed and 286 zygotes analyzed. No difference was detected between replicates, and the results were pooled. Fisher's exact test was used to determine effect of treatment. More zygotes had 2 PN after fertilization with semen from bull A (47/67 = 70%), compared with that from bull B (16/71 = 23%; P < 0.0001) using nonirradiated sperm. Irradiation of sperm significantly increased the fraction of zygotes with 2 PN from bull A (61/70 = 87%; P = 0.03), but decreased the 2 PN fractions in zygotes fertilized with sperm from bull B (2/75 = 3%; P = 0.0002). Ideally, to avoid straw variation, semen should have been pooled and divided into groups before irradiation. The variation between straws might explain the higher fertilization rates in bull A using irradiated sperm compared with nonirradiated sperm. In conclusion, there appear to be differences in fertilizing ability between bulls after irradiation of frozen–thawed sperm. This could be due to suboptimal DNA packaging, which made sperm from bull B more susceptible to radiation-induced damage. The potential irradiation-induced increase in DNA fragmentation in sperm from bull B compared with that from bull A might delay or prevent the formation of the 2 PN. Further studies are needed to investigate differences in fertilization and early embryonic development using sperm with intact or damaged DNA.
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Ojaghi, Mina, Chinju Johnson, Guilherme Rizzoto, John Kastelic, and Jacob C. Thundathil. "Content and activity of the testis-specific isoform of angiotensin-converting enzyme are reduced in frozen–thawed bull spermatozoa." Reproduction, Fertility and Development 30, no. 11 (2018): 1575. http://dx.doi.org/10.1071/rd17219.
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Sperm cryopreservation and thawing reduces fertility and alters the content and function of various sperm proteins. Previously, we reported that a testes-specific isoform of angiotensin-converting enzyme (tACE) was required for capacitation of bovine spermatozoa. The aim of the present study was to determine effects of sperm cryopreservation and thawing on the content, activity and localisation of tACE in bovine spermatozoa. Relative median fluorescence intensity (flow cytometry) was greater (P < 0.01), tACE content (110 kDa protein) in sperm proteins was higher (P < 0.01) and there was greater tACE enzyme activity (mean (±s.e.m.) 0.16 ± 0.01 vs 0.06 ± 0.02 U mL−1; P < 0.01) in fresh versus frozen–thawed spermatozoa (n = 6 bulls). In fresh spermatozoa, tACE was immunolocalised in the acrosomal and principal piece regions of the sperm head and tail respectively. However, in frozen–thawed spermatozoa, there were four patterns of localisation: most frozen–thawed spermatozoa (64%) had fluorescence in the acrosomal ridge, whereas in 17% and 9% of spermatozoa the signal was limited to the post-acrosomal region and the equatorial segment respectively; in the remainder (10%), there was no signal. We conclude that cryopreservation and thawing decrease the content and activity of tACE and cause it to be translocated to other parts of the sperm head.
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Knobbe, M., M. Reese, M. Kaproth, I. Dobrinski, and H. Galantino-Homer. "Assessing cryocapacitation in frozen-thawed bovine sperm from a commercial bull stud." Theriogenology 70, no. 3 (August 2008): 586. http://dx.doi.org/10.1016/j.theriogenology.2008.05.024.
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GUNJI, Satomi, and Seizo HAMANO. "Effect of β-mercaptoethanol Treatment on Capacitation of Frozen-Thawed Bull Spermatozoa." Nihon Chikusan Gakkaiho 72, no. 9 (2001): 329–36. http://dx.doi.org/10.2508/chikusan.72.9_329.
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Ahmad, Ejaz, Misbah Hanif, Muhammad Saleem Akhtar, Tanveer Ahmad, Tanveer Hussain, Adeel Ahmad, Zia Ur Rehman Farooqi, and Masroor Ellahi Babar. "Effect of adenosine 5' triphosphate (ATP) on frozen thawed buffalo bull spermatozoa." Cryobiology 97 (December 2020): 260. http://dx.doi.org/10.1016/j.cryobiol.2020.10.048.
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Biniová, Zuzana, Luděk Stádník, Martina Doležalová, and Jaromír Ducháček. "Effect of thawing method on bull sperm survival in ejaculates frozen in 4 ml and 8 ml volumes." Czech Journal of Animal Science 63, No. 10 (September 27, 2018): 399–407. http://dx.doi.org/10.17221/117/2018-cjas.
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The aim of this study was to evaluate the effect of different thawing protocols (slow (P1), medium (P2), and fast (P3)) on percentage of motile sperm (MOT) and percentage of sperm cells with intact membranes (INT) in Holstein (4 bulls; 72 samples) and Czech Fleckvieh (4 bulls; 72 samples) semen frozen-thawed in 4 ml and 8 ml volumes. MOT was analysed in fresh semen, as well as immediately after thawing (T0) and 30 min after thawing (T30). INT was analysed using hypoosmotic swelling test (HOS test) in fresh ejaculate (FE), after diluting (DE), and at T0. The differences between FE parameters and frozen-thawed ejaculate parameters, expressing changes that occur during cryopreservation, were calculated. Apart from the effect of thawing protocol, the effect of breed and the effect of quality of FE expressed by MOT immediately after collecting were evaluated, too. Unlike thawing of semen cryopreserved in straws (0.25 and 0.5 ml), thawing using the slow protocol (P1) was the most appropriate (P < 0.05) for both observed volumes. There were found significantly higher MOT in the volume of 8 ml in both T0 and T30 and in the volume of 4 ml in T30 in samples thawed using P1 and P2. MOT in T0 was significantly affected by breed in samples frozen in 8 ml and in T30 in samples frozen in 4 and 8 ml. There were found no significant differences in INT in all reported volumes, however decrease of INT during cryopreservation was affected by breed.
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Tshabalala, M. M., K. A. Nephawe, M. L. Mphaphathi, C. M. Pilane, and T. L. Nedambale. "21 Effect of egg yolk extracted low-density lipoprotein on cryopreserved Nguni bull semen." Reproduction, Fertility and Development 31, no. 1 (2019): 136. http://dx.doi.org/10.1071/rdv31n1ab21.
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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<0.05. Sperm motility and membrane integrity were significantly higher (P<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<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.
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Thys, M., A. Van Soom, J. Dewulf, T. Rijsselaere, and A. de Kruif. "113 EFFECT OF SPERM COATING ON THE QUALITY OF BOVINE FROZEN - THAWED SPERMATOZOA." Reproduction, Fertility and Development 18, no. 2 (2006): 164. http://dx.doi.org/10.1071/rdv18n2ab113.
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The substantial decrease of sperm quality after cryopreservation remains an important issue in the artificial insemination industry. Sperm coating with Triladyl® (Minitübe, Tiefenbach, Germany) during ejaculation can preserve sperm characteristics and oocyte penetrating capacity of fresh bovine spermatozoa stored in egg yolk diluent for up to 6 days (De Pauw et al. 2003 Theriogenology 59, 1109–1122). Since collecting semen in a tube containing egg yolk-Tris extender (sperm coating) limits the period of contact between spermatozoa and seminal plasma, the present experiment was conducted to assess if this slightly adjusted method of sperm collection could also have a significant effect on bovine sperm quality after cryopreservation. Semen of five young Holstein Friesian bulls was collected by means of an artificial vagina connected to an empty tube (Group 1; five ejaculates per bull) or a tube containing 4 mL of an egg yolk-Tris extender (Groups 2 and 3; each five ejaculates per bull). The semen samples of Group 1 were conventionally diluted in straws (60 × 106 sperm/mL), frozen, and stored in liquid nitrogen. The samples of Group 3 were centrifuged, and after removing diluent and seminal plasma, the sperm pellet was conventionally diluted and processed. The samples of Group 2 were processed without removal of the supernatant. After thawing each ejaculate was analyzed for average path velocity (VAP), beat cross frequency (BCF), and progressive motility (PROG) using CASA (Minitübe, Tiefenbach, Germany). Furthermore, the membrane integrity of each sample was evaluated using fluorescent SYBR®–14/PI staining (BD Biosciences, Erembodegem, Belgium). All parameters were compared among the three groups of sperm using univariate analysis of variance (SPSS 12.0; SPSS, Inc., Chicago, IL, USA). No significant differences could be observed among the three groups for all of the evaluated sperm characteristics (Table 1). A significant effect of the bull could be determined for all analyzed parameters (P ≤ 0.02), except for the percentage of moribund cells. Nevertheless, the group-bull interaction was never statistically significant. Coating bovine sperm with an egg yolk-Tris extender during ejaculation cannot prevent the substantial deterioration of the spermatozoa that occurs during freezing and thawing since this method of sperm collection does not significantly influence the motility parameters or the membrane integrity after thawing. Table 1. VAP, BCF, PROG, and percentage of membrane-intact, dead, and moribund spermatozoa for the three groups of sperm This research was supported by IWT (no. IWT/020727).
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Seshoka, M. M., M. L. Mphaphathi, K. S. Mafolo, M. Nkadimeng, Z. C. Raphalalani, N. L. Kanuya, and T. L. Nedambale. "38 QUAIL EGG YOLK IN CITRATE EXTENDER IS SUITABLE FOR CRYOPRESERVATION OF NGUNI BULL SEMEN." Reproduction, Fertility and Development 28, no. 2 (2016): 149. http://dx.doi.org/10.1071/rdv28n2ab38.
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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.
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Mphaphathi, M. L., M. M. Seshoka, F. V. Ramukhithi, Z. C. Raphalalani, T. R. Netshirovha, A. Maqhashu, N. L. Kanuya, M. B. Raito, J. P. C. Greyling, and T. L. Nedambale. "15 QUANTIFICATION OF BULL SPERM TRAITS AS ASSESSED BY COMPUTER-ASSISTED SEMEN ANALYSIS AND THE RELATIONSHIP TO PREGNANCY RATE FOLLOWING CONTROLLED BREEDING." Reproduction, Fertility and Development 29, no. 1 (2017): 115. http://dx.doi.org/10.1071/rdv29n1ab15.
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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 < 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 < 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.
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Dhami, A. J., Tapasvi M. Patel, and DV Chaudhari. "Correlations Amongst Functional and Morphological Attributes and Oxidative Markers of Fresh and Cryopreserved Semen of Gir and Murrah Bulls." INDIAN JOURNAL OF VETERINARY SCIENCES AND BIOTECHNOLOGY 15, no. 03 (March 9, 2020): 24–29. http://dx.doi.org/10.21887/ijvsbt.15.3.7.
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This study was undertaken during the winter season on healthy mature Gir cattle and Murrah buffalo bulls (n=3 each). The semen samples (6 ejaculates/bull, total 36 ejaculates) collected in the morning using artificial vagina were evaluated for routine seminal attributes, including acrosomal and plasma membrane integrity. The samples were then diluted @ 100 million sperm/ml with tris fructose yolk glycerol extender without and with sericin @ 0.1, 0.25, 0.5 and 1.0% (w/v), filled in French mini-straws, and frozen in LN2 using biofreezer as per standard freezing protocol. Straws were thawed in water bath at 37°C for 30 sec and evaluated for post-thaw quality, viz., motility, viability, morphology, acrosome integrity and plasma membrane integrity (HOST). Lipid peroxidation (malondialdehyde - MDA production) and activities of enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) were assessed as oxidative markers in seminal plasma of freshly diluted and frozen-thawed semen samples. Sericn at 0.5% level significantly (p less than 0.01) improved the post-thaw sperm quality with reduced oxidative stress in both the species. The breed-wise correlation coefficients (r) among sperm quality attributes and oxidative markers were studied in fresh and frozen-thawed semen of each species, and also for fresh with frozen-thawed semen. The findings revealed significant interrelationships amongst most of the attributes of fresh as well as post-thawed semen and also of fresh semen attributes with those of cryopreserved semen including oxidative markers in both the species. Sperm motility estimation in fresh, pre-freeze and post-thawed semen was a legitimately good indicator of quality of spermatozoa at various steps of semen processing/freezing, and its fertilizing potential. Thus, the sperm motility, HOS test and either MDA or SOD/GPx activity alone may be used as valuable and practical tools for routine assessment of bovine semen quality considering significant correlations found between them.
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Chen, Xiaoli, Yonggui Wang, Huabin Zhu, Haisheng Hao, Xueming Zhao, Tong Qin, and Dong Wang. "Comparative transcript profiling of gene expression of fresh and frozen–thawed bull sperm." Theriogenology 83, no. 4 (March 2015): 504–11. http://dx.doi.org/10.1016/j.theriogenology.2014.10.015.
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Maulana, T., and S. Said. "Kinematics motility of frozen-thawed X and Y sperm of Sumba Ongole bull." IOP Conference Series: Earth and Environmental Science 387 (December 5, 2019): 012030. http://dx.doi.org/10.1088/1755-1315/387/1/012030.
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Thundathil, J., J. Gil, A. Januskauskas, B. Larsson, L. Söderquist, R. Mapletoft, and H. Rodriguez-Martinez. "Premature capacitation and fertility of frozen-thawed bull semen used in artificial insemination." Theriogenology 51, no. 1 (January 1999): 351. http://dx.doi.org/10.1016/s0093-691x(99)91910-6.
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Mehmood, A., M. Anwar, and SM Saqlan Naqvi. "Capacitation of Frozen Thawed Buffalo Bull (Bubalus bubalis) Spermatozoa with Higher Heparin Concentrations." Reproduction in Domestic Animals 42, no. 4 (August 2007): 376–79. http://dx.doi.org/10.1111/j.1439-0531.2006.00794.x.
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BUDWORTH, PAUL R., RUPERT P. AMANN, and PHILIP L. CHAPMAN. "Relationships Between Computerized Measurements of Motion of Frozen-Thawed Bull Spermatozoa and Fertility." Journal of Andrology 9, no. 1 (January 2, 1988): 41–54. http://dx.doi.org/10.1002/j.1939-4640.1988.tb01007.x.
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Seshoka, M. M., M. L. Mphaphathi, and T. L. Nedambale. "53 COMPARISON OF 4 DIFFERENT CRYOPROTECTANTS ON FREEZING NGUNI BULL SPERMATOZOA EVALUATED BY COMPUTER-AIDED SPERM ANALYSIS." Reproduction, Fertility and Development 26, no. 1 (2014): 140. http://dx.doi.org/10.1071/rdv26n1ab53.
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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 < 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.
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Pribenszky, C. S., M. Molnar, L. Solti, J. Dengg, and J. Lederer. "98 THE EFFECT OF HIGH HYDROSTATIC PRESSURE ON THE MOTILITY OF FRESH AND FROZEN-THAWED BULL SEMEN." Reproduction, Fertility and Development 17, no. 2 (2005): 199. http://dx.doi.org/10.1071/rdv17n2ab98.
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Previously, we reported that a sublethal shock, high hydrostatic pressure (HHP), significantly improves the post-thaw survival of frozen mouse blastocysts, presumably from the induction of shock proteins (Pribenszky et al. 2004 Reprod. Fert. Dev. 16, 181). Others reported that HSP90 in spermatozoa decreased substantially after freezing (Huang et al. Theriogenology 51, 1007–1016; Cao Wen-Lei et al. 2003 Asian J. Androl. 5, 43–46). We now report the effect of HHP on motility of the fresh bull semen to determine whether sperm survives in an altered pressure environment, and to compare post-thaw motility of HHP-treated frozen bull semen with controls. The survival rates were compared by chi-square test. Expt 1: Semen of one bull was diluted to a sperm concentration of 8 × 107/mL with AndroMed extender (MiniTüb, Tiefenbach, Germany). Diluted sperm was loaded into 0.25-mL straws at 25°C. Each straw was cut in half. One demi-straw was heat-sealed and exposed to HHP, and sperm in the companion demi-straw served as a control. Experiments were replicated eight times for each pressure/time treatment. Progressive motility was assessed independently by light microscopic investigation by two individuals. The treatment groups were: 10 MPa for 30, 60, 90, or 120 min; 30 MPa for 30, 60, 90, 120, or 510 min; 50 MPa for 30, 60, or 90 min; 70 MPa for 30, 60, or 90 min; and 90 MPa for 30, 60, 90, 120, or 510 min. The average motility of the control samples ranged from 75 to 90%, while the average motility of the pressurized samples ranged between 55 (90 MPa/120 min) to 84% (10 MPa/30 min). The groups of 30 MPa/510 min and 90 MPa/510 min exhibited significantly lower motility compared to the other pressurized groups (27% and 33%, respectively; P < 0.05). Expt 2: Semen was collected from two bulls with poor sperm freezability. Semen was diluted as described for the first experiment, loaded into straws, and assigned to one of 4 treatment groups. Half the straws from each bull were exposed to 90 MPa/30 min, 90 MPa/90 min, 30 MPa/30 min, or 30 MPa/90 min, and then cryopreserved. Controls consisted of straws that were cryopreserved without pressure treatment. Cryopreservation steps were 60 min equilibration at 5°C, followed by 10 min at −110°C, and then plunging into liquid nitrogen. Straws were thawed in a 35°C water-bath for 30 s. Each treatment and control group was replicated 8 times (8 samples per bull). The average post-thaw motility was significantly superior with pressure pre-treatment in each of the pressurized groups compared to the samples frozen without previous pressurization (P < 0.001) (Bull I: 2–3% without pressurization vs. 17–33% with pressurization; Bull II: 0% without pressurization vs. 21–35% with pressure pre-treatment). Among the pressure/time parameters used, 30 MPa/90 min proved significantly superior (33 and 35%; P < 0.05) for each of the bulls. Expt. 2 clearly demonstrates the beneficial effect of a previous pressure treatment on post-thaw motility of bull semen cryopreserved in our experiment. Further investigations are needed, including samples from different bulls, different freezing protocols, and the biological background of the process. This work was supported partly by NKFP 4/040/2001.
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Puglisi, Roberto, Roberta Vanni, Andrea Galli, Donatella Balduzzi, Katia Parati, Graziella Bongioni, Gabriella Crotti, et al. "In vitro fertilisation with frozen–thawed bovine sperm sexed by flow cytometry and validated for accuracy by real-time PCR." Reproduction 132, no. 3 (September 2006): 519–26. http://dx.doi.org/10.1530/rep.1.01173.
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The methodologies used for cytometric sorting of fresh spermatozoa never allowed a clear resolution of sexual chromosomes of frozen–thawed semen. To devise a novel method for the production of bovine predefined sexed embryos using frozen–thawed semen, sorting efficiency of different protocols was studied using a new quantitative real-time PCR method to verify the purity of sexed semen. To this aim, after Percoll separation, frozen–thawed samples were stained at different temperatures and concentrations of Hoechst 33342 using a short-incubation time. The concentration of Hoechst 33342 of 500 μg/ml at a temperature of 37 °C provided good and stable fluorescence signals. Preventing the sperm clustering by adding 0.6% BSA in the 90% Percoll fraction led to X-bearing sperms purity of 91±2%. Thereafter, sorted sperms were used for in vitro fertilisation (IVF). Despite the lower cleavage rates reported in the sorted groups when compared with the control groups (40 vs 48%, P<0.01), blastocyst formation in the sorted and control groups was not different (27 vs 24% of the cleaved respectively). The PCR analysis of 30 blastocysts confirmed 26 embryos to be correctly sexed (87%). Transfer of two embryos per recipient into six synchronised heifers resulted in four pregnancies. Two abortions occurred at day 60, while two pregnancies went to term delivering two female calves. In conclusion, high purity and repeatabilityof sorting was obtained with frozen–thawed bull semen that was subsequently used for IVF giving rise toviable embryos and offspring. In addition, real-time PCR revealed to be an optimal support for these studies, providing a rapid and reliable estimation of flowcytometric efficiency.
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Ardon, Florencia, and Susan S. Suarez. "Cryopreservation increases coating of bull sperm by seminal plasma binder of sperm proteins BSP1, BSP3, and BSP5." REPRODUCTION 146, no. 2 (August 2013): 111–17. http://dx.doi.org/10.1530/rep-12-0468.
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Artificial insemination with frozen semen allows affordable, worldwide dissemination of gametes with superior genetics. Nevertheless, sperm are damaged by the cryopreservation process. Elucidating the molecular effects of cryopreservation on sperm could suggest methods for improving fertility of frozen/thawed semen. This study was undertaken to examine the effect of cryopreservation on the coating of sperm by binder of sperm (BSP) proteins in seminal plasma. BSP proteins are secreted by the seminal vesicles and coat the surface of sperm by partially intercalating into the outer leaflet of the sperm plasma membrane. The BSP proteins are known to play roles in the formation of the oviductal sperm storage reservoir and in sperm capacitation. We investigated the effects of cryopreservation on the sperm BSP protein coat using Bovipure to separate live sperm from extended semen and then assaying the amounts of BSP proteins on sperm using quantitative western blotting with custom-made antibodies against unique sequences of each BSP protein. Greater amounts of all three BSP proteins (BSP1, BSP3, and BSP5) were detected on frozen/thawed sperm than on fresh sperm. Furthermore, the reduction of BSP3 from 15 to 13 kDa in mass, which occurs during incubation of sperm under mild capacitating conditions, was enhanced by cryopreservation. We concluded that freezing alters the BSP protein coating on sperm, which could account in part for reduced fertility of cryopreserved semen samples.
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Dinkins, Michael B., and Benjamin G. Brackett. "Chlortetracycline staining patterns of frozen-thawed bull spermatozoa treated with β-cyclodextrins, dibutyryl cAMP and progesterone." Zygote 8, no. 3 (August 2000): 245–56. http://dx.doi.org/10.1017/s0967199400001040.
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Efforts to achieve complete chemical definition of media used for in vitro capacitation of bovine spermatozoa including removal of heparin purified from porcine intestinal mucosa are presented. Fluorescent staining with chlortetracycline (CTC), known to reflect changes coincident with sperm capacitation in certain species, was studied following treatments of frozen-thawed bull spermatozoa with β-cyclodextrins, dibutyryl cAMP (dbcAMP) and progesterone in comparison with heparin. The CTC staining patterns (F, B and AR) were confirmed to correlate with known conditions that effectively prepare cryopreserved bull spermatozoa for fertilisation in vitro. In the absence of glucose, the routinely employed heparin-containing capacitating medium caused an increase in spermatozoa displaying the AR pattern. Both progesterone (100 μM) and dbcAMP (0.01–0.1 mM) were able to increase the proportion of B pattern stained sperm cells more than after exposure to control (mDM) conditions without a significant reduction in motility. Exposure to either dbcAMP or β-cyclodextrins was accompanied by an increase in proportions of spermatozoa displaying the AR pattern over those seen in controls. Exposure to β-cyclodextrins did not increase the proportion of B pattern stained spermatozoa. Comparison of spermatozoa from two bulls revealed differential responses of spermatozoa from different males to treatments with heparin and progesterone. In vitro fertilisation results demonstrated that previously cryopreserved bull spermatozoa could be capacitated in chemically defined conditions devoid of heparin or other biological components.
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Frers, L. G., J. Hepburn, K. Hogan, C. Parminter, L. Mc Gowan, R. Vishwanath, S. Beaumont, and R. McDonald. "269 THE USE OF A NEW EXTENDER FOR STORING FRESH BOVINE SEMEN FOR LONG PERIODS." Reproduction, Fertility and Development 22, no. 1 (2010): 291. http://dx.doi.org/10.1071/rdv22n1ab269.
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Processing bovine semen in fresh long life extender for use over 3 to 4 days after collection is a widely used technique in New Zealand (Shannon and Vishwanath 1995 Anim. Reprod. Sci. 39, 1-10). Advantages include greater use of valuable sires, transport without liquid nitrogen, and the possibility of more efficient use of sexed sorted semen. The new extender (Ext. A) also has the advantage of containing no egg yolk. This study compares this new long-life extender (Ext. A) with an existing product (Ext. B) and frozen/thawed semen. Semen from 12 different bulls was diluted to a concentration of 8 × 106 mL-1 and gradually cooled to 16°C. All samples were held at ambient temperature in the dark and motility was evaluated over a storage period of 4 days comparing the extenders. In this part of the trial Ext. A maintained motility better than Ext. B (P = 0.001) during the 4-day storage period (24 h: 90 v. 70%; 96 h: 85 v. 50%). The second part of the trial compared the conception rates (CR) in cows from the use of fresh long-term-extended semen and frozen/thawed semen. On 19 farms, 8546 cows were inseminated with fresh semen stored for 1 to 3 days and 7280 cows were inseminated with frozen semen. The overall CR at 7 to 8 weeks for the 19 farms was 73.7%. On 18 farms within the same farming group, 8498 cows received frozen semen and the CR was 71.1%. Pregnancy results were 2.6% (P = 0.001) higher CR at scanning in herds where fresh semen was used compared with the farms where only frozen/thawed semen was used (73.7 v. 71.1%). In the third part of the trial, semen from 4 different bulls were extended to 1 × 106 mL-1 in Ext. A and held at ambient temperature for 6 days prior to use for IVF. Our lab standard frozen/thawed bull semen was used as a control. Table 1 shows that semen held at ambient temperature in Ext. A for 6 days produced a similar percentage of transferable quality embryos to our IVP control frozen/thawed semen (26.9 v. 25.7%). We conclude that preserved bovine semen in fresh long-life extender for several days offers some advantages in AI and IVP programs compared with frozen semen. Table 1.Fresh semen extender (Ext.A) compared with frozen semen in IVP We appreciate the assistance of Liberty Genetics Ltd.
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Garcia, O. S., R. S. Ferro, J. M. D. Bezerra, and M. S. Sales. "271 EFFECT OF BULL ON IN VITRO FERTILIZATION OF BOVINE OOCYTES." Reproduction, Fertility and Development 22, no. 1 (2010): 292. http://dx.doi.org/10.1071/rdv22n1ab271.
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The main objective of this work was to evaluate the effect of the bull on IVF of bovine oocytes. These oocytes were obtained from ovaries collected in the slaughterhouse and selected for their maturation process in TCM-199 culture medium with 20% estrus cow serum, 10 IU of eCG, and 1 mg mL-1 gentamicin. The oocytes were cultivated for 20 to 24 hours in 39.0°C culture incubator and moist environment with 5% CO2. For the oocyte insemination, frozen/thawed semen of 8 bulls was used by means of swim-up capacity procedure according to Parrhit (1989) and then motility, sperm concentration, and strengh were evaluated. In all cases, 25 to 30 oocytes, previously selected according to cumulus oophorus density were artificially inseminated with 1 × 106 spermatozoids mL-1. The total of selected oocytes by bull for this work was: 5028 (1), 1247 (2), 2888 (3), 650 (4), 1529 (5), 699 (6), 984 (7), and 1250 (8). The results were evaluated according to the average of oocytes matured in vitro and to embryonic division 48 hours after AI. In order to evaluate the effects by bull, the statistical examination of chi-square proportion comparison was used. The average values and standard deviation (SD) motility were 57.0% ± 13.7; 53.2% ± 11.3; 51.2% ± 8.20; 40.9% ± 5.0; 48.1% ± 4.92; 33.5% ± 4.42; 53.5% ± 11.1 and 40.0% ± 8.9 for bulls 1 to 8, respectively. There was a highly significant difference (P < 0.001) in relation to the percentage of division: 46, 42, 34, 18, 30, 31, 30, and 17% among bulls 1 to 8, respectively, in addition to variability among these bulls. The results obtained confirm the effect of the bull on conception rate. The conclusion is that frozen/thawed semen with 50% motility can increase the percentage of embryonic division.