Made available in DSpace on 2014-06-11T19:35:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-26Bitstream added on 2014-06-13T20:46:11Z : No. of bitstreams: 1 crespilho_am_dr_botffmvz.pdf: 1224891 bytes, checksum: 5f6cd3bb0ea028b0690772b9a9bf8493 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo do estudo foi comparar a efetividade de três diluidores empregados para criopreservação e refrigeração do sêmen bovino em relação aos padrões de motilidade, integridade de membrana plasmática e acrossomal, índice de peroxidação lipídica e fertilidade nos programas de inseminação artificial em tempo-fixo (IATF). No Trabalho científico número 1 foi comparado a viabilidade e fertilidade pós-descongelação proporcionada pelos diluidores Tris-frutose (TRIS, Controle) e Botu-Bov® (BB), ambos contendo 20% de gema de ovo como fonte de lipoproteínas, frente à diluição em Botu- Bov®-Lecitina de Soja (meio BB-L) apresentando 1% de lecitina em substituição ao produto de origem animal. No Trabalho 2 foram avaliados os mesmos diluentes quando utilizados para a refrigeração do sêmen bovino por 48 horas a 5°C. Já no Trabalho número 3 foi avaliada a taxa de concepção na inseminação artificial (C/IA) proporcionada pelo sêmen bovino refrigerado por 24 horas em meio Botu-Bov® em comparação ao sêmen convencionalmente criopreservado no mesmo diluidor. Os meios TRIS e BB a base de gema de ovo foram mais efetivos na manutenção da viabilidade espermática pósdescongelação, conferindo melhores resultados de C/IA (P<0,05) em relação ao meio BBL. No entanto, quando utilizado o sêmen na forma líquida e refrigerado (Trabalho número 2) foi observada uma maior proteção contra o estresse oxidativo proporcionado pelo diluidor a base de lecitina de soja, resultando em maior probabilidade de prenhez quando comparado às amostras refrigeradas em TRIS ou BB, alcançando índice de concepção similar ao obtido com o sêmen congelado. A utilização do sêmen bovino refrigerado por 24 horas levou ao aumento da C/IA de vacas submetidas a IATF quando comparado ao sêmen congelado em meio Botu-Bov®. Conclui-se que embora a lecitina de soja represente... / The aim of this study was to compare three different extenders used for cryopreservation of bovine semen, based on the results obtained during the cooling storage and post-thaw evaluation for motility patterns, integrity of plasmatic and acrossomal membranes, lipid peroxidation rate, as well as conception rate after fixed-time artificial insemination (FTAI). In Paper.1, the efficiency of Tris-Fructose extender (TRIS, control group), Botu-Bov® extender (BB), both containing 20% of egg yolk, and Botu-Bov®-Lecithin extender (BBL), which has 1% of soy lecithin instead of egg yolk, were compared in cryopreservation of bovine semen. In Paper.2, ejaculates from different bulls were cooled to 5°C for 48 hours using the same extenders of Paper.1. In Paper.3 the fertility trial was conducted either with frozen-thawed semen or cooled semen for 24 hours in the BB extender. The egg yolk extenders, TRIS and BB, demonstrated significant differences on the viability and the fertility of frozen-thawed bovine semen when compared to BB-L (P<0.05). However, the use of lecithin instead of egg yolk on semen extender resulted in a greater protection against oxidative stress; moreover, this extender improved the conception rates, reaching the results obtained in FTAI programs with frozen-thawed semen. The use of cooled bovine semen at 5°C for 24 hours improves the conception rate of Nelore cows submitted to FTAI. Although soy lecithin is an interesting alternative source of phospholipids in the elaboration of chemically defined extenders and decrease the risk of microbiological contamination, the egg yolk semen extenders are more effective in preserving the viability and fertility of frozen-thawed bovine semen. However, there was a higher production of free radicals in cooled semen with the use of egg yolk based extenders, resulting in lower conception rates when compared to frozen-thawed... (Complete abstract click electronic access below)

Orientador: Frederico Ozanam Papa / Banca: José Antonio Dell'Aqua Junior / Banca: Eunice Oba / Banca: Alicio Martins Junior / Banca: Rubens Paes de Arruda / Resumo: O objetivo do estudo foi comparar a efetividade de três diluidores empregados para criopreservação e refrigeração do sêmen bovino em relação aos padrões de motilidade, integridade de membrana plasmática e acrossomal, índice de peroxidação lipídica e fertilidade nos programas de inseminação artificial em tempo-fixo (IATF). No Trabalho científico número 1 foi comparado a viabilidade e fertilidade pós-descongelação proporcionada pelos diluidores Tris-frutose (TRIS, Controle) e Botu-Bov® (BB), ambos contendo 20% de gema de ovo como fonte de lipoproteínas, frente à diluição em Botu- Bov®-Lecitina de Soja (meio BB-L) apresentando 1% de lecitina em substituição ao produto de origem animal. No Trabalho 2 foram avaliados os mesmos diluentes quando utilizados para a refrigeração do sêmen bovino por 48 horas a 5°C. Já no Trabalho número 3 foi avaliada a taxa de concepção na inseminação artificial (C/IA) proporcionada pelo sêmen bovino refrigerado por 24 horas em meio Botu-Bov® em comparação ao sêmen convencionalmente criopreservado no mesmo diluidor. Os meios TRIS e BB a base de gema de ovo foram mais efetivos na manutenção da viabilidade espermática pósdescongelação, conferindo melhores resultados de C/IA (P<0,05) em relação ao meio BBL. No entanto, quando utilizado o sêmen na forma líquida e refrigerado (Trabalho número 2) foi observada uma maior proteção contra o estresse oxidativo proporcionado pelo diluidor a base de lecitina de soja, resultando em maior probabilidade de prenhez quando comparado às amostras refrigeradas em TRIS ou BB, alcançando índice de concepção similar ao obtido com o sêmen congelado. A utilização do sêmen bovino refrigerado por 24 horas levou ao aumento da C/IA de vacas submetidas a IATF quando comparado ao sêmen congelado em meio Botu-Bov®. Conclui-se que embora a lecitina de soja represente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to compare three different extenders used for cryopreservation of bovine semen, based on the results obtained during the cooling storage and post-thaw evaluation for motility patterns, integrity of plasmatic and acrossomal membranes, lipid peroxidation rate, as well as conception rate after fixed-time artificial insemination (FTAI). In Paper.1, the efficiency of Tris-Fructose extender (TRIS, control group), Botu-Bov® extender (BB), both containing 20% of egg yolk, and Botu-Bov®-Lecithin extender (BBL), which has 1% of soy lecithin instead of egg yolk, were compared in cryopreservation of bovine semen. In Paper.2, ejaculates from different bulls were cooled to 5°C for 48 hours using the same extenders of Paper.1. In Paper.3 the fertility trial was conducted either with frozen-thawed semen or cooled semen for 24 hours in the BB extender. The egg yolk extenders, TRIS and BB, demonstrated significant differences on the viability and the fertility of frozen-thawed bovine semen when compared to BB-L (P<0.05). However, the use of lecithin instead of egg yolk on semen extender resulted in a greater protection against oxidative stress; moreover, this extender improved the conception rates, reaching the results obtained in FTAI programs with frozen-thawed semen. The use of cooled bovine semen at 5°C for 24 hours improves the conception rate of Nelore cows submitted to FTAI. Although soy lecithin is an interesting alternative source of phospholipids in the elaboration of chemically defined extenders and decrease the risk of microbiological contamination, the egg yolk semen extenders are more effective in preserving the viability and fertility of frozen-thawed bovine semen. However, there was a higher production of free radicals in cooled semen with the use of egg yolk based extenders, resulting in lower conception rates when compared to frozen-thawed... (Complete abstract click electronic access below) / Doutor

The aim of the current study was to determine if frozen-thawed bull semen can be treated with the acidic buffer MES (2-[N- morpholino] ethanesulfonic acid) without any detrimental effect on the motility, plasma membrane, acrosomal membrane and longevity of sperm. Frozen bull semen was obtained from a local co-operative. The semen was frozen in 0.25 mL French straws at a concentration of 80 x 106 sperm cells per millilitre. Semen of two different batches from ten bulls of four different breeds was used in this study. Three frozen semen straws of each batch were thawed at 38° C for 25 seconds. The thawed semen was pooled and then split into two aliquots. The one aliquot was used as control, whilst the other was exposed to MES treatment. The motility, plasma membrane integrity, acrosomal membrane integrity and longevity of sperm were evaluated. The effect of MES on motility was minimal as only the percentage of aberrantly motile sperm increased two hours after treatment. Although no effect on the plasma membranes were observed, it can be assumed that some damage did occur due to the fact that the acrosomal membranes were affected significantly. No significant effect was found for longevity of sperm between the control and treated samples, but a significant effect was found for both the control and treated samples over time. Although the detrimental effects caused by MES treatment would render some sperm unable to fertilise an oocyte, it is likely that a sufficient portion of sperm would survive the treatment. It is probable that this treatment would also be effective in frozen-thawed buffalo semen. The following step would be to treat semen of footand-mouth disease positive bulls with MES to establish if treatment with MES will be effective in inactivating foot-and-mouth disease virus in semen of infected bulls. Copyright / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2008. / Production Animal Studies / unrestricted

Orientador: Frederico Ozanam Papa / Banca: João Carlos Pinheiro Ferreira / Banca: Alício Martins Junior / Resumo: A despeito das inúmeras variáveis que influenciam direta e indiretamente a fertilidade das fêmeas bovinas, a qualidade das amostras seminais exerce um papel importante na determinação das taxas de concepção dos programas de inseminação artificial. Os objetivos dessa pesquisa foram comparar a efetividade de dois diluidores de criopreservação de sêmen bovino no processamento de amostras seminais apresentando diferentes concentrações espermáticas em relação aos índices de congelabilidade determinados laboratorialmente (Experimento I) e as taxas de concepção proporcionadas por cada metodologia quando utilizada em programas de inseminação artificial em tempo fixo (IATF) em bovinos (Experimento II). No Experimento I foram utilizados 14 ejaculados de diferentes touros da raça Nelore. Cada ejaculado foi fracionado em oito alíquotas iguais, submetidas a criopreservação com os diluidores Tris-gema de ovo-frutose (meio TRIS) e MKA nas concentrações de 12, 25, 50 e 100 milhões de espermatozóides totais por mililitro de meio, formando oito grupos experimentais em função das variáveis diluidor e concentração. As amostras foram descongeladas a 46 ºC por 20 segundos, avaliando-se os padrões de motilidade através do método computadorizado (CASA), integridade de membrana plasmática (IMP), resistência ao teste de termorresistência rápido (TTR) e taxa de recuperação e IMP após seleção espermática pela técnica de swim-up. Para o Experimento II foram selecionados sete touros utilizados no Experimento I, obtendo-se um ejaculado de cada animal por eletroejaculação...(Resumo completo, clicar acesso eletrõnico abaixo) / Abstract: Although there are many variables which directly or indirectly influence female bovine fertility, the quality of sperm samples plays a important role in the determination of conception rates in artificial insemination programs. The aim of the present study was to compare the efficiency of two bovine semen extenders for sperm freezing with different spermatic concentrations in the freezability determined by lab tests (Experiment I), and conception rates after fixed time artificial insemination (FTAI; Experiment II). In Experiment I 14 ejaculates of different Nelore bulls were used. Each ejaculate was splitsampled in to eight equal parts and then submitted to cryopreservation with Tris-egg yolk fructose (TRIS) and MKA extenders, at concentrations of 12, 25, 50 and 100 millions spermatozoa per milliliter forming eight experimental groups. The samples were thawed at 46 ºC for 20 seconds, and the following parameters were evaluated: sperm motility and movement (by computer-assisted semen analysis - CASA), sperm membrane integrity (SMI), resistance to the fast thermoresistance test (TT), recovery rate and sperm membrane integrity after sperm selection through swim-up technique. Seven of 14 bulls used in Experiment I were selected for Experiment II, and semen was collected from each of the animals by electroejaculation. The seven ejaculates obtained were mixed (semen pool) and cryopreserved, thus forming eight experimental groups according to the freezing extenders and sperm concentrations/straws: TRIS 12, 25, 50 and 100, and MKA 12, 25, 50 and 100...(Complete abstract, click electronic address below) / Mestre

MSCAGR / Department of Animal Science / Preservation of semen is an important process to ensure that semen quality is sufficient for use in assisted reproductive technologies. This study evaluated the effectiveness of three different extenders to preserve bull semen stored under different conditions, as an alternative to frozen-thawed semen straws used for artificial insemination. Semen samples were collected from two Nguni bulls using an electro-ejaculator and transported to the laboratory at 37 °C for evaluation. Pooled semen was first aliquoted into three extenders namely Triladyl, Ham’s F10 and M199 at a dilution ratio of 1:4 (semen:extender), and then stored at controlled room temperature 24 °C. Secondly pooled semen was aliquoted into four groups of Ham’s F10 extender and diluted at a ratio of 1:4, then stored at 24 °C, 17 °C, 12 °C and 5 °C respectively. Sperm motility rates were analysed after 0, 24, 48 and 72 hours. Morphology, viability and sperm DNA fragmentation were analysed after 72 hours. The study was replicated four times and data was analysed by ANOVA. Triladyl had higher sperm viability rate and total motility rate for 72 hours (P<0.01). However, Ham’s F10 had higher progressive motility rate as compared to the other extenders. There was no significant difference (P<0.01), in the viability rate between Ham’s F10 and M199. No significant difference was also observed in total sperm abnormalities (absent tails, coiled tails and bent tails), except for reacted acrosomes (P>0.05), between the two Nguni bulls. Lower temperatures than 24 °C influenced sperm motility and viability in Ham’s F10. There was no significant difference in sperm DNA fragmentation rates (P<0.01), between all the four storage temperatures which indicated that temperature did not have an influence on sperm DNA fragmentation. In conclusion, bull semen can be preserved in Triladyl or Ham’s F10 and M199 culture media stored at 24 °C and stay alive for 72 hours. Triladyl proved to be the best suitable extender showing higher sperm viability and total motility rates as compared to Ham’s F10 and M199. Lower temperatures than 24 °C noticeably decreased sperm motility and viability in Ham’s F10 culture medium.

Made available in DSpace on 2014-06-11T19:29:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-26Bitstream added on 2014-06-13T19:38:31Z : No. of bitstreams: 1 crespilho_am_me_botfmvz.pdf: 507525 bytes, checksum: a1c92f545e77b50838125fbea8dfddd6 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A despeito das inúmeras variáveis que influenciam direta e indiretamente a fertilidade das fêmeas bovinas, a qualidade das amostras seminais exerce um papel importante na determinação das taxas de concepção dos programas de inseminação artificial. Os objetivos dessa pesquisa foram comparar a efetividade de dois diluidores de criopreservação de sêmen bovino no processamento de amostras seminais apresentando diferentes concentrações espermáticas em relação aos índices de congelabilidade determinados laboratorialmente (Experimento I) e as taxas de concepção proporcionadas por cada metodologia quando utilizada em programas de inseminação artificial em tempo fixo (IATF) em bovinos (Experimento II). No Experimento I foram utilizados 14 ejaculados de diferentes touros da raça Nelore. Cada ejaculado foi fracionado em oito alíquotas iguais, submetidas a criopreservação com os diluidores Tris-gema de ovo-frutose (meio TRIS) e MKA nas concentrações de 12, 25, 50 e 100 milhões de espermatozóides totais por mililitro de meio, formando oito grupos experimentais em função das variáveis diluidor e concentração. As amostras foram descongeladas a 46 ºC por 20 segundos, avaliando-se os padrões de motilidade através do método computadorizado (CASA), integridade de membrana plasmática (IMP), resistência ao teste de termorresistência rápido (TTR) e taxa de recuperação e IMP após seleção espermática pela técnica de swim-up. Para o Experimento II foram selecionados sete touros utilizados no Experimento I, obtendo-se um ejaculado de cada animal por eletroejaculação... / Although there are many variables which directly or indirectly influence female bovine fertility, the quality of sperm samples plays a important role in the determination of conception rates in artificial insemination programs. The aim of the present study was to compare the efficiency of two bovine semen extenders for sperm freezing with different spermatic concentrations in the freezability determined by lab tests (Experiment I), and conception rates after fixed time artificial insemination (FTAI; Experiment II). In Experiment I 14 ejaculates of different Nelore bulls were used. Each ejaculate was splitsampled in to eight equal parts and then submitted to cryopreservation with Tris-egg yolk fructose (TRIS) and MKA extenders, at concentrations of 12, 25, 50 and 100 millions spermatozoa per milliliter forming eight experimental groups. The samples were thawed at 46 ºC for 20 seconds, and the following parameters were evaluated: sperm motility and movement (by computer-assisted semen analysis - CASA), sperm membrane integrity (SMI), resistance to the fast thermoresistance test (TT), recovery rate and sperm membrane integrity after sperm selection through swim-up technique. Seven of 14 bulls used in Experiment I were selected for Experiment II, and semen was collected from each of the animals by electroejaculation. The seven ejaculates obtained were mixed (semen pool) and cryopreserved, thus forming eight experimental groups according to the freezing extenders and sperm concentrations/straws: TRIS 12, 25, 50 and 100, and MKA 12, 25, 50 and 100...(Complete abstract, click electronic address below)

Bislang wurden Bullen in der Milchrinderzucht anhand des Töchterleistungsvergleiches bewertet, was zur Folge hatte, dass die Zuchtwertschätzung erst nach fünf Jahren vorlag (REENTS UND REINHARDT, 2007). Durch die 2010 eingeführte genomische Zuchtwertschätzung werden Bullenkälber bereits in den ersten Lebensmonaten geprüft und die erfolgreichen Bullen ab einem Alter von 12 Monaten zur Spermaproduktion von den Besamungsstationen eingesetzt. Zu bedenken ist dabei, dass Jungbullen unter 2 Jahren lediglich ein durchschnittliches Ejakulatvolumen von 2-4 ml aufweisen und die Spermienkonzentration deutlich unter der von Altbullen liegt. Es müssen daher entweder mehr Jungbullen zum Einsatz kommen oder die Besamungsportionen müssen in ihrer Spermienkon-zentration reduziert werden. Eine am Institut für Nutztiergenetik Mariensee ent-wickelte Methode, der Sperm-Intra-Fallopian-Transfer (SIFT®), ermöglicht es, minimal dosierte Spermaportionen unchirurgisch in den Eileiter zu übertragen (GROSSFELD ET AL., 2011B). Ein wesentlicher Vorteil dieser Methode ist die Re-duzierung des Besamungsvolumens unter Beibehaltung des Volu-men/Mengenverhältnisses. Zielsetzung dieser Arbeit war es, eine in Volumen und Spermienzahl reduzierte Spermaportion neu zu konfektionieren und ein angepasstes Kühl- sowie Gefrierprotokoll zu entwickeln. Hierbei sollten vergleichbare Auftauqualitäten wie in einer üblichen Besamungsportion erreicht werden. Die Qualität des Spermas wurde dabei sowohl unter Laborbedingungen als auch in Testbesamungen an zufällig ausgewählten Färsen und Kühen evaluiert. In ersten Screeningversuchen wurde zunächst ein Tiefgefrierverfahren für gering dosierte Verpackungssysteme (Nanostraw) mit einem Volumen bis 50 µl entwickelt. Dabei sollten vergleichbare Auftauqualitäten üblicher Besamungs-portionen erreicht werden. Es wurden sechs verschiedene Kühlkurven (A-D, D+ und E) entwickelt. Dabei zeigte sich, dass aufgrund der Stickstoff-volumeneinströme ein Haltepunkt bei -8°C in keiner der Einfrierkurven für kleine Volumina zu etablieren war. Dies ist u.a. auf die bauartbedingten Vorgaben des verwendeten Einfrierautomaten zurückzuführen. Ebenso wurde der Zusatz von 7,5% Glyzerin im Verdünner bei dem Gefrierprotokoll D+ aufgrund der Datenlage des Vorversuchs für den Hauptversuch verworfen. Die Proben wurden bullenindividuell erhoben, zur besseren statistischen Absicherung jedoch für die Auswertung zusammengefasst. Aus den Screeningversuchen erschien das Kryokonservierungsprotokoll D+ für den Nanostraw unter Laborbedingungen am geeignetsten und wurde im Thermoresistenztest, der den Bedingungen im Hinblick auf die Temperatur im Uterus bzw. Eileiter ähnlich ist, getestet. Weiterhin fand ein Vergleich der spermatologischen Qualitätsparameter nach dem Auftauen zwischen der Kontroll- und der Nanostrawgruppe mit der Kühlkurve D+ im Thermoresistenztest statt. Im praktischen Teil der Arbeit wurde die Methode SIFT® zunächst anhand eines Versuchs mit geschlechtsdifferenziertem Frischsamen praktiziert. Dabei wurden 20 Färsen mittelseiner Prid®α 1, 55g Progesteron-Spirale und zehn Kühe mit einem Ovsynch Programm synchronisiert. In einem weiteren Besamungsversuch konnte das Sperma aus den Nanostraws verwendet werden, das vorher kryokonserviert worden ist. Es wurden nur spontan brünstige Färsen und Kühe in den Eileiter besamt. Die Untersuchungen führten zu folgenden Ergebnissen: 1) In den Screeningversuchen zeigte sich, dass der Anteil motiler Spermien signifikant (p≤0,05) abhängig von der Gefriergeschwindigkeit der Kühlkurve war. Der höchste Anteil motiler Spermien aus dem Nanostraw war in Kühlkurve D+ mit einem Anteil von 52,9% zu verzeichnen. Eine signifikante Zunahme des Anteils membranintakter Spermien von Kurve A zu B und D zu D+ war zu beobachten. Die Hälfte der Nanostrawspermien zeigten in der Kühlkurve D+ eine intakte Membranintegrität. Die Kontrollspermien lagen bei 56,9%. In Kurve D+ zeigten 66,6% der Spermien aus dem Nanostraw keine morphologischen Veränderungen, in der Kontrollgruppe waren dies 75,4%. Die Änderung der Glyzerinkonzentration im Verdünnungsmedium zeigte keine signifikante Verbesserung der Spermaqualitätsparameter nach dem Auftauen. 2) Im Thermoresistenztest erreichten die Spermien aus dem Nanostraw in allen gemessenen Parametern die Mindestanforderungen an die Spermaqualität nach Rath et al. (2009). So belief sich der Anteil membranintakter Spermien aus dem Nanostraw nach dreistündiger Inkubation bei 37°C auf 56,4%, die Kontrollstrawspermien lagen bei einem Anteil von 67,2%. Dabei zeigte der Faktor Bulle einen signifikanten Effekt (p≤0,01) auf den Anteil motiler und progressiver Spermien sowie den Anteil PI-negativer Spermien. Mögliche Effekte des Bullen auf die Qualität und Befruchtungsfähigkeit der Spermien nach dem Auftauen zeigten andere Versuchsanstellungen und Forschungshypothesen (JANUSKAUSKAS ET AL., 1996, KATHIRAVAN ET AL., 2011). 3) In dem ersten Besamungsversuch wurden 20 Färsen und zehn Kühen geschlechtsdifferenziertes Sperma mit der Methode SIFT® in den Eileiter über-tragen. 37,5% der Färsen und 33% der Kühe wurden tragend. Dabei konnte ein bullenindividueller dosisabhängiger Effekt festgestellt werden. 50% der Kühe, die mit 500.000 Samenzellen inseminiert wurden und 16% der Kühe, die mit 250.000 Samenzellen besamt wurden, wurden tragend. 4) Im Besamungsversuch mit dem kryokonservierten Sperma aus dem Na-nostraw wurden sechs spontan brünstige Rinder mittels SIFT®-Spermien in den Eileiter übertragen. Alle Tiere wurden als Kälber für die Follikelpunktion in einem anderen Versuch verwendet. Fünf Tiere wurden mit dem Bullen A besamt. 40% der Tiere wurden tragend. Lediglich ein Tier wurde mit dem Bullen B besamt. Das Trächtigkeitsergebnis war negativ. Die Kühe wurden mit dem Bullen A besamt, dieser erzielte eine Trächtigkeitsrate von 33%.