Orientador: Marco Antonio Alvarenga / Banca: Marco Antonio Alvarenga / Resumo: Em eqüinos existe um grande interesse em se desenvolver e aprimorar técnicas de inseminação artificial que possibilitam a obtenção de bons índices de fertilidade utilizando um baixo número de espermatozóides. O presente trabalho teve como objetivos avaliar: 1) a viabilidade, in vitro, do sêmen congelado de garanhões após a diluição em fluido folicular pré-ovulatório de éguas; 2) a população espermática no oviduto de éguas, utilizando diferentes técnicas de inseminação; 3) a fertilidade de éguas após a utilização de diferentes técnicas de inseminação artificial, utilizando um baixo número de espermatozóides; 4) a reação inflamatória na cavidade peritoneal após as inseminações intrafoliculares e intraperitoneais; 5) averiguar o efeito da presença de espermatozóides dentro do folículo pré-ovulatório, na capacidade de fertilização do oócito. No experimento 1 foram realizadas análises laboratoriais da viabilidade espermática, de sêmen congelado de garanhões sem diluição (GI), diluído em Kenney (GII) e diluído em fluido folicular (GIII). Observou-se que porcentagem de espermatozóides com membrana plasmática íntegra não diferiu significativamente entre os grupos (p>0,05). Também não foi observado diferença estatística (p>0,05) entre os grupos na porcentagem de espermatozóides vivos com membrana acrossomal íntegra. A motilidade total foi significativamente inferior (p<0,05) no grupo em que o sêmen foi diluído em fluido folicular (GIII) apenas no primeiro momento, logo após a diluição (T0). A motilidade progressiva foi significativamente maior (p<0,05) no grupo diluído com Kenney (GII) do que nos demais grupos em T0 e três horas após (T3). A velocidade espermática ao longo de uma trajetória média (VAP) foi significativamente maior (p<0,05) em GIII do que em GI nos momentos T0, após uma (T1) e duas horas (T2) da diluição. / Abstract: The objective of the present study was to evaluated the viability, in vitro, of stallion frozen semen after dilution in mare's pre-ovulatory follicular fluid; assess sperm population in mares's oviduct using differents artificial insemination techinics; verify the fertility of of differents artificial insemination techinics in mares with low sperm number; assess if mares develop an inflamatory reaction in the peritoneal cavity after intraperitoneal and intrafollicular insemination and to verify if the presence of sperm in pre-ovulatory follicle could interfere on oocyte fertilization capacity. In experiment 1, frozen-thawed semen viability was evaluated without dilution (GI) and after dilution with Kenney or Mare's pre-ovulatory follicular fluid. The percentage of sperm with plasmatic membrane integrity was not different between the groups (p>0,05). No statistical difference (p>0,05) was observed between the groups in percentage of live sperm with intact acrosome. Total motility (MT) was significantly lower (p<0,05) in follicular fluid group (GIII) just in the first moment (T0). Progressive motility was significantly higher (p<0,05) in group diluted with Kenney (GII) than the other groups in T0 and 3 hours after (T3). Average path velocity (VAP) was significantly higher (p<0,05) in GIII than GI in moments T0, T1 and T2. Straight line velocity (VSL), curvilinear velocity (VCL) and Beat cross frequence (BCF) were significantly lower (p<0,05) in GI in moments T0, T1 and T2, when compared with GIII. No statistical difference (p>0,05) were observed in amplitud of lateral head (ALH), straightness (STR) and percentage of sperm with linear (LIN) and rapid (RAP) movement between GI and GIII in any moment. In T3 no statistical difference was observed (p>0,05) between all groups in any motility parameters analyzed. / Doutor

Orientador: Frederico Ozanam Papa / Resumo: O presente estudo objetivou a colheita do sêmen de garanhões nos haras, diluição e transporte destas amostras em dois sistemas de refrigeração (Equitainerâ e Max-Semen Expressâ) para os centros especializados onde foram submetidos ao processo de congelação. No Experimento I foi utilizado 01 ejaculado de 12 garanhões de diferentes raças. Após a colheita os ejaculados foram divididos em duas alíquotas: uma submetida ao processo de congelação convencional, utilizando o meio diluidor Botu-Crioâ e outra diluída com Botu-Semenâ e armazenada em Equitainerâ durante 24h. Após a refrigeração o sêmen foi centrifugado e ressuspendido com Botu-Crioâ e submetidas ao processo de congelação. As amostras descongeladas foram analisadas pelo CASA e a integridade de membrana plasmática pelo uso de sondas fluorescentes. Os resultados do Experimento I mostraram que não houve diferença nos parâmetros avaliados (motilidade total, progressiva e integridade da membrana plasmática) entre a metodologia convencional e a congelação de sêmen após 24h de refrigeração (p>0,05). No Experimento II, foi utilizado 01 ejaculado de 11 garanhões, os quais foram diluídos e armazenados em dois sistemas de transporte (Equitainerâ e Max-Semen Expressâ) durante 24h. As amostras foram congeladas, conforme descrito no Experimento I utilizando-se a associação do glicerol com dimetilacetamida, dimetilformamida e metilformamida. Após a descongelação a análise dos parâmetros espermáticos demonstrou a superioridade do Equitainerâ em relação ao Max-Semen Expressâ (p<0,05). A metilformamida associada ao glicerol apresentou resultados superiores aos demais crioprotetores utilizados . O teste de fertilidade comparou amostras congeladas de dois garanhões (A e B) pelos métodos: convencional e pós-refrigeração em Equitainer por 24h.... (Resumo complet, clicar acesso eletrônico abaixo) / Abstract: The aim of the present study was to develop a new method to freeze equine semen. Semen was collected at the stallion farm, diluted, divided into two samples and cooled at Equitainer® or Max-Semen Express®, then transported to an equipped laboratory for freezing. On Experiment I used one ejaculate from each of 11 stallions from different breeds. The ejaculates were divided into two parts: one was frozen following a regular protocol and the other was diluted with Botu-Semen® and stored at Equitainer® for 24 hours. Afterwards, cooled semen was centrifuged and ressuspended with Botu-Crio® and then frozen. After thawing motility was evaluated by CASA and by fluorescent probes for plasma membrane integrity evaluation. Based on these results of Experiment I concluded that there was no difference between total motility, progressive motility and plasma membrane integrity when comparing the two methods for freezing semen (p>0,05). At Experiment II one ejaculate from each of 11 stallions was collected, diluted and stored either an Equitainer® or in a Max-Semen Express® for 24 hours. The samples were frozen as described on Experiment I using the association of glycerol with dimethylformamide, dimethylacetamide and methylformamide. After thawing the results suggested that the Equitainer® was better than Max-Semen Express® in maintaining sperm viability (p>0,05). The association of metilformamida and glycerol was superior to the others. Fertility trials compared the two methods: conventional and cooled/frozen semen. Ten mares were inseminated in a total of 40 cycles; the mares were monitorated every day by ovarian ultrasonography. Ovulation was induced using 10mg EPE (equine pituitary extract) intravenously when a follicle of 35mm of diameter was detected. Inseminations were performed twice, before and after ovulation with 350x106 spermatozoa/mL toward the tip of the horn.... (Complete abstract, access undermentioned electronic address) / Mestre

O melhoramento genético ovino carece de uma maior conexão genética entre os rebanhos, o que pode solucionado pelo uso mais intensivo do sêmen congelado. Entretanto, com os níveis tecnológicos atualmente disponíveis o sêmen congelado apenas pode ser empregado com eficácia diretamente no útero, dependendo de sincronização de estros e mão de obra especializada para as inseminações via laparoscopia. Uma outra alternativa é o uso de sêmen congelado com 200 milhões de espermatozóides por dose com inseminações efetuadas cerca de doze horas após a identificação do estro, preconizado para a inseminação artificial dos ovinos na Noruega pelos próprios produtores. O presente ensaio consta de uma adaptação local deste modelo com o objetivo geral de verificar a viabilidade de uso da inseminação cervical superficial com sêmen ovino congelado numa dose de 0,25 ml contendo 200 x 106 de espermatozóides em palhetas de 0,50 ml após cio natural, empregando baixo uso de insumos e mão de obra semi-qualificada. As observações foram procedidas em duas propriedades no Rio Grande do Sul, incluindo 1419 ovelhas das raças Merino, Ideal e Texel. Os resultados obtidos indicaram que as diferenças nas taxas de não retorno entre carneiros poderá ser minimizada através da utilização de outros métodos de análise e seleção de carneiros mais férteis após os procedimentos de congelamento/descongelamento, e, adicionalmente que a constatação de uma taxa de não retorno entre 25-35% nas distintas condições investigadas, permitem inferir que os sistemas investigados podem ser utilizados para interligar geneticamente rebanhos via uso de sêmen congelado. / A sheep improvement program depends on genetic connexion among flocks, which could be done by extensive use of frozen semen. However, nowadays-available technology just recommends the employment of frozen semen directly inside the uterine ambient through laparoscopy. Alternatively, there is a model recommended for Norway producers including frozen semen with 200 millions of sperms used in cervical superficial inseminations up to twelve hours from oestrus detection. The present essay is an local adaption of this system aiming to verify the viability of the employment of frozen semen for sheep reproduction in a volume of 0,25 ml, with 200 x 106 sperms in 0,50 ml straws, after natural oestrus, using low input resource and semi-qualified artificial insemination technicians. The observations were done in the two properties located at Rio Grande do Sul, including 1419 ewes from Merino, Ideal and Texel breeds. The results obtained indicate that the differences in non return rates among rams could be minimized by the employment of other methods of ram selection before the freezing procedures, and additionally the observed 25-35% non return rates in the distinct conditions investigated, permits to infer that the tested system could be useful to connect genetically the flocks using frozen semen.

Made available in DSpace on 2014-08-20T14:37:56Z (GMT). No. of bitstreams: 1 dissertacao_rafael_tonieto.pdf: 295673 bytes, checksum: cde6d39f839653a9d49de2dfbef8ef62 (MD5) Previous issue date: 2008-10-24 / The low pregnancy rates obtained with artificial insemination using frozen ram semen make its routine use unfeasible. As the cryopreservation process causes injuries in the sperm cells, the development of extenders that include state-of-the art cryoprotectant solutions is justified. This study tested the inclusion of low density lipoprotein (LDL) and trehalose in extenders for freezing ram semen, by evaluating parameters of post-thawing semen quality. In the first experiment, 3 treatments were compared: Tris including 20% egg yolk and 5% glycerol (T1); Tris including 10 mM trehalose (T2); and T1 including 50 mM trehalose (T3). Sperm motility did not differ across treatments (P > 0.05), but T2 presented higher proportion of sperm cells having membrane integrity (P < 0.05). In the second experiment, 4 treatments were compared: Tris including 20% egg yolk (T1); T1 including 5% glycerol (T2); T1 including 100 mM trehalose (T3); and t1 including 100 mM trehalose and 5% glycerol (T4). Sperm motility and membrane integrity were higher for T2, T3 and T4 than for T1 (P < 0.05), but acrossome integrity did not differ among treatments (P > 0.05). In the third experiment, four treatments were compared: Tris including 20% egg yolk and 100 mM trehalose; and T1 with the replacement of egg yolk by LDL including 5% glycerol (T2); 100 mM trehalose (T3); and 100 mM trehalose and 5% glycerol (T4). Sperm motility and membrane integrity were higher for T2 and T3 than for the other treatments (P < 0.05), but there was no further difference among the LDL treatments (P > 0.05). Acrossome integrity did not differ across treatments (P > 0.05). Therefore, the inclusion of LDL and trehalose as cryoprotectants in extenders for frozen ram semen was associated with improvement in post-thawing sperm motility and membrane integrity. / Em ovinos, as baixas taxas de prenhez obtidas com inseminação artificial com sêmen congelado inviabilizam o seu uso como rotina. Provocando o processo de criopreservação lesões nos espermatozóides, o desenvolvimento de diluentes que incluam crioprotetores de excelência se justifica. Este trabalho testou a inclusão da lipoproteína de baixa densidade (LDL) e da trealose em diluentes para congelamento de sêmen ovino, a partir da avaliação de parâmetros de qualidade seminal pós-descongelamento. No primeiro experimento, foram comparados 3 tratamentos: Tris com 20% de gema de ovo e 5% de glicerol (T1); Tris com 100 mM de trealose (T2); e T1 com 50 mM de trealose (T3). A motilidade não diferiu entre os tratamentos (P > 0,05), mas o T2 apresentou maior proporção de gametas com membranas íntegras (P < 0,05). No segundo experimento, foram comparados 4 tratamentos: Tris com 20% de gema de ovo (T1); T1 com 5% de glicerol (T2); T1 com 100 mM de trealose (T3); e T1 com 100 mM de trealose e 5% de glicerol (T4). A motilidade e a integridade da membrana espermática do T2, T3 e T4 foram superiores a do T1 (P > 0,05), mas a integridade do acrossoma não diferiu (P > 0,05) em todos os tratamentos. No terceiro experimento, foram comparados 4 tratamentos: Tris com 20% de gema de ovo e 110 mM de trealose (T1); Tris com 8% de LDL, incluindo 5% de glicerol (T2); Tris com 8% de LDL, incluindo 110 mM de trealose (T3); Tris com 8% de LDL, incluindo 110 mM de trealose e 5% de glicerol (T4). A motilidade dos espermatozóides para o T2 e para o T3 foram superiores aos demais tratamentos (P < 0,05), mas, com relação à integridade de membrana, não houve diferença entre os tratamentos com LDL (P > 0,05). A integridade do acrossoma não diferiu entre os tratamentos (P > 0,05). Portanto, o uso de LDL e trealose como crioprotetores foi associado com melhorias na motilidade e na integridade da membrana espermática, no sêmen ovino, após o descongelamento.

Four experiments were conducted to evaluate the viability and fertility of bovine spermatozoa encapsulated in microcapsules and microgels. In Experiment I, one of two morphologically distinct sperm types i.e. marker or unmarked bull spermatozoa (100 x 10⁶ sperm/bull) were encapsulated in protamine sulfate microcapsules and simultaneously inseminated with the reciprocal sperm type unencapsulated. Insemination of both sperm types unencapsulated served as a control. Accessory sperm embedded in the zona pellucida were counted and morphologically classified 6 to 7 d post insemination. From microencapsulated inseminates, accessory sperm populations did not increase over the unencapsulated controls, but contributed 25.7% of the accessory sperm population. ln Experiment 2, an in vitro study was performed to evaluate the maintenance of viability for bovine spermatozoa encapsulated in PIPES, HEPES, or saline microgels. Neat semen was pooled from five bulls (50 x 10° sperm/bull), encapsulated in alginate microgels, and incubated at 37 C for 8 h. The unencapsulated control displayed greater maintenance of viability for percent intact acrosomes and motility when compared to all treatments. By 8 h incubation, PlPES and HEPES were not significantly different, but demonstrated greater maintenance of viability when compared to saline microgel treatments. In Experiment 3, PIPES microgels were heterospermically inseminated with equal numbers (20 x l0⁶ sperm/bull) of frozen-thawed marker bull and normal bull spermatozoa as explained in Experiment 1. Microencapsulated treatments contributed significantly lower numbers of accessory sperm when compared to unencapsulated controls. In Experiment 4, one of the two morphologically distinct sperm types (20 x l0⁶ frozen-thawed sperm/bull) were encapsulated in protamine sulfate microcapsules and the reciprocal sperm type was encapsulated in PIPES microgels. A total of 21 accessory sperm were recovered from 30 embryos which demonstrates the ability of microencapsulated spermatozoa to fertilize an oocyte. / Master of Science

Includes bibliographical references (leaves 232-257). / xv, 257 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aims to determine if the compatible solutes (proline, glycine betaine, and trehalose), the epididymal compounds (taurine, hypotaurine and inositol) or the antioxidants (carnosine and ascorbic acid) in tris-citrate based diluents could improve the post-thaw survival and/or fertility of ram spermatazoa. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Science, 1996?

Made available in DSpace on 2014-06-11T19:35:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-27Bitstream added on 2014-06-13T21:08:05Z : No. of bitstreams: 1 villaverde_aisb_me_botfmvz.pdf: 995611 bytes, checksum: 95781868451ff6d5037fde5a7435a721 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo deste estudo foi comparar a eficiência das técnicas de inseminação artificial intra-vaginal (IAIV) e intra-uterina (IAIU) com sêmen congelado/descongelado em gatos domésticos através da taxa de prenhez. O ejaculado de dois gatos adultos foi colhido com vagina artificial e o sêmen avaliado para motilidade (CASA), morfologia espermática e integridade de membrana plasmática. Após diluição em meio TRIS/OEP (4% de glicerol), as amostras foram envasadas em palhetas francesas de 0,25 mL (25 x 106 de espermatozóides móveis) as quais, após 20 minutos à 5ºC, permaneceram por 15 minutos em vapor de nitrogênio líquido, sendo posteriormente mergulhadas. Para cada IA, quatro palhetas do mesmo gato foram descongeladas por 12 segundos à 46ºC e centrifugadas a 250xg por 8 minutos. O pellet ressuspendido em 100 æL do meio foi analisado como descrito anteriormente. Nas gatas, o estro e a ovulação foram induzidos com 100 UI de eCG e, após 84 horas, 100 UI de hCG. Após 30 horas da indução da ovulação, as gatas foram inseminadas pelo método intra-uterino ou intra-vaginal, com o sêmen obtido do gato A ou B (n=8 gatas para cada método de IA). Para a análise estatística os testes de Tukey e de Wilcoxon foram utilizados para as variáveis de qualidade espermática, enquanto o teste de Fisher foi usado para comparar os métodos de IA, estabelecendo diferença significativa quando p<0,05. Embora tenha sido observada uma queda acentuada de motilidade, integridade de acrossomo e integridade de membrana plasmática nas amostras seminais descongeladas de ambos os gatos, um índice de prenhez de 75% foi alcançado quando utilizado o método de IA intra-uterino, contra 0% com o método intra-vaginal. / The aim of this study was to compare the efficiency of the intravaginal and the intrauterine artificial insemination methods using domestic cat frozen/thawed semen by determining the pregnancy rate. The sperm was collected from two tom cats using an artificial vagina and the samples were assessed for motility (CASA), sperm morphology and plasma membrane integrity. After dilution with TRIS/OEP (4% of glycerol), the sperm samples were loaded into 0.25 mL straws (25 x 106 of sperm cells presenting motility) which, after 20 minutes under 5ºC, were placed in liquid nitrogen vapour for 15 minutes, being afterwards immersed. For each AI, four straws from the same male were thawed during 12 seconds at 46ºC and centrifuged at 250xg for 8 minutes. The pellet was ressuspended in 100 æL of the extender and analyzed as described above. In the queens, estrus and ovulation were induced using 100 UI of eCG and, after 84 hours, 100 UI of hCG. After 30 hours from the ovulation induction, the females were inseminated by the intrauterine or by the intravaginal methods, using the semen obtained by male A or B (n=8 females for each AI method). For statistical analysis the Tukey test and the Wilcoxon test were used for the sperm quality results, while the Fisher test was used to compare the two AI methods, with p<0.05 taken as significant. Although a pronounced decrease in motility, acrosome integrity and plasma membrane integrity were observed for the sperm samples from both cats, a pregnancy rate of 75% was achieved when using the intrauterine AI method, against 0% with the intravaginal AI method.

Made available in DSpace on 2014-08-20T14:37:56Z (GMT). No. of bitstreams: 1 dissertacao_karina_lemos_goularte.pdf: 361604 bytes, checksum: c1cf54e456adc029afb9c9829a7d4c67 (MD5) Previous issue date: 2009-03-11 / The expansion of artificial insemination (AI) programs with frozen ram semen has been limited by the need of intrauterine semen deposition and by the inconsistency of the association between the conventional methods used to evaluate semen quality and in vivo fertility. An alternative to improve the efficiency of AI programs would be the profiling of the protein content of the seminal plasma to search for fertility markers. The objective of the present study was to study the protein profile of ram seminal plasma and to evaluate its association with parameters of frozen semen quality. Ejaculates were split in two samples. The first sample was frozen in two extenders: T1 (Tris + egg yolk + glycerol); and T2 (Tris + egg yolk + threalose). The second sample was submitted to unidimensional electrophoresis to identify proteins in the seminal plasma. The presence of the identified proteins was associated with parameters of semen quality: sperm motility and membrane integrity, both prefreezing and post-thawing, and post-thawing acrosome integrity. Post-thawing sperm motility did not differ (P > 0.05) for T1 (18.6 ± 2.3) and T2 (23.4 ± 2.7). Post-thawing sperm membrane integrity was similar (P > 0.05) for T1 (12.6 ± 1.4) and T2 (15.3 ± 1.7). There was also no difference (P > 0.05) between T1 and T2 with regard to postthawing acrosome integrity (23.0 ± 1.5 e 21.3 ± 1.8, respectively). An intra-ram analysis identified 17 proteins associated with the evaluated parameters: 10 of them presented similar associations for distinct rams. In the inter-ram analysis, an 11 kDa band was associated with lower pre-freezing sperm membrane integrity, a 24 kDa band was related to reduced post-thawing sperm motility and membrane integrity, and a 45 kDa band was associated with lower pre-freezing sperm membrane integrity (P < 0.05). Thus, those three protein factors would be potential markers for ram infertility with frozen semen because their absence was associated with improved semen quality, regardless of individual ram effects. / A expansão de programas de inseminação artificial (IA) em ovinos com sêmen congelado tem sido limitada pela necessidade de deposição intrauterina do sêmen e pela associação inconsistente entre as técnicas convencionais de avaliação da qualidade do sêmen e a fertilidade in vivo. Uma das alternativas para incrementar a eficiência de programas de IA seria o mapeamento do conteúdo protéico do plasma seminal, em busca de marcadores para fertilidade. O objetivo do presente trabalho foi estudar o perfil protéico do plasma seminal de machos ovinos e avaliar a sua associação com a qualidade de amostras de sêmen congelado. Os ejaculados dos machos ovinos foram divididos em duas alíquotas. A primeira foi utilizada para criopreservação, em dois diluentes: D1 (tris+gema de ovo+glicerol); e D2 (tris+gema de ovo+trealose). A segunda alíquota foi usada para a busca de proteínas do plasma seminal através da eletroforese unidimensional. A presença ou ausência das proteínas foi associada com parâmetros de qualidade seminal: motilidade e integridade da membrana espermática, ambas pré-congelamento e pósdescongelamento, e integridade de acrossoma pós-descongelamento. A motilidade espermática pós-descongelamento não diferiu (P > 0,05) entre D1 (18,6 ± 2,3) e D2 (23,4 ± 2,7). A integridade da membrana espermática pós-descongelamento foi semelhante (P > 0,05) para D1 (12,6 ± 1,4) e D2 (15,3 ± 1,7). Também não houve diferença (P > 0,05) entre D1 e D2 quanto à integridade do acrossoma pósdescongelamento (23,0 ± 1,5 e 21,3 ± 1,8, respectivamente). A análise intra-machos identificou 17 bandas proteicas associadas com os parâmetros avaliados, sendo que 10 destas associaram-se da mesma maneira, em diferentes machos. Na análise inter-machos, a banda com 11 kDa foi associada com menor integridade da membrana espermática pré-congelamento, a banda com 24 kDa foi associada com a redução na motilidade e na integridade da membrana pós-descongelamento e a banda com 45 kDa foi associada com menor integridade da membrana précongelamento (P < 0,05). Portanto, essas três bandas protéicas seriam potenciais marcadores para infertilidade com sêmen congelado, pois sua ausência foi associada com incremento na qualidade seminal, independentemente do efeito dos machos doadores de sêmen.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / During the 2005 and 2006 southern hemisphere natural breeding season, 104 Criollo mares were used to evaluate deep frozen semen fertility from 5 Criollo stallions. FR4 was used as extender (Nutricell, Brazil) with 5% glycerol (GLY) or 5% dimethylformamid (DMF) as cryoprotectants. Semen was loaded in 0.5mL straws (100x106 spermatozoa/straw). Mares were examined every other day by ultrasonography until detection of ≥30mm diameter follicles, when hCG was injected (2500UI i.v.). After 24 hours, mares were examined daily in Experiment I, and every 6 hours, in experiment II. Insemination with frozen semen was performed deep in the uterine horn ipsilateralis to the dominant follicle. Controls were inseminated with fresh semen in the body of the uterus. Only one estrus period per mare was used. Pregnancy diagnosis through ultrasonography was performed on the 15th day postovulation. In experiment I, pregnancy rates were 11.9% (5/42) and 62.5% (20/32), respectively for DMF and fresh semen (P<0.0001). In experiment II, pregnancy rates were 40% (4/10), 10% (1/10) and 70% (7/10), respectively for DMF, GLY (P>0.05) and fresh semen (P<0.0001). It was showed that GLY and DMF can be used for frozen of Criollo Stallion s semen. In this context DMF brought better results. / Durante a estação reprodutiva natural de 2005 e 2006 do hemisfério sul, 104 éguas Crioulas foram utilizadas para avaliar a fertilidade do sêmen congelado de cinco garanhões Crioulos. Como diluente usou-se FR4 (Nutricell, Brasil) com 5% de glicerol (GLI) ou 5% de dimetilformamida (DMF) como crioprotetores. O sêmen foi envasado em palhetas de 0,5mL (100x106 espermatozóides/palheta). Examinou-se as éguas em dias alternados por ultra-sonografia transretal para detecção de folículos ≥30mm de diâmetro, quando recebiam hCG (2500UI i.v.). Apos 24h eram examinadas diariamente no Experimento I e a cada 6h no Experimento II. As inseminações com sêmen congelado eram realizadas no fundo do corno ipsislateralis ao folículo dominante. Nas controle, com sêmen fresco no corpo uterino. Utilizou-se um ciclo estral/animal. O diagnóstico de gestação foi conduzido por ultra-sonografia no 15o dia pós-ovulação. No experimento I, a taxa de prenhez foi 11,9% (5/42) e 62,5% (20/32), respectivamente para DMF e sêmen fresco (P<0,0001). No experimento II, a taxa de prenhez foi 40% (4/10), 10% (1/10) e 70% (7/10), respectivamente para DMF, GLI (P>0,05) e sêmen fresco (P<0,0001). Demonstrou-se que o GLI e a DMF podem ser utilizados como crioprotetores para o congelamento do sêmen de garanhões Crioulos. Neste contexto, a DMF apresentou melhores resultados.

<p>In recent years conservation of minor livestock breeds has been faced with numerous challenges attributed to decreasing national herd sizes, as well as differences in reproduction and growth. One such minor swine breed, the Large Black pig (LB), is increasingly attractive to small farmers due to their foraging abilities and carcass characteristics. Therefore, the LB pigs have been used in niche pork production systems which market pasture-raised pork products. The LB breed is critically endangered, maintaining a registered breeding population of less than 400 animals, with increasing prevalence of inbreeding and genetic drift. Therefore, the LB breed could benefit from a genetic importation to increase genetic diversity in a national herd with rapidly decreasing animal numbers. A genetic importation would require frozen semen to be brought in from another country for use in breeding U.S. pigs. Frozen-thawed semen (FTS) presents challenges for swine due to the reduced motile sperm cells which negatively impacts fertility. Therefore, the present study evaluated the utilization of FTS in a genetic importation for the LB pig. </p><p>A genetic importation occurred in 2016 where semen from the United Kingdom was used on various farms in the U.S. but resulted in zero piglets born. Therefore, 16 LB sows were donated to Purdue University for research into improving estrous and ovulation synchronization to facilitate FTS utilization. Four breeding replicates were performed where following 14 days of Matrix feeding, OvuGel® was administered at 144 h following last Matrix feeding (LMF) or 96 h in post-weaned sows and two FTS inseminations occurring at: 30 and 36 h, 17 and 23 h, 24 and 30 h, and 24 and 32 h after OvuGel® for replicates 1-4, respectively. Approximately 2.64±0.3 billion motile sperm cells per insemination were utilized in replicates 1-3 with American LB FTS, with replicate 4 utilizing 0.34±0.03 billion motile sperm cells of imported FTS. Follicle diameter (<i>P</i>=0.260), ovulation within 48 h of OvuGel® (<i>P</i>=0.411), and weight prior to breeding (<i>P</i>=0.681) did not influence conception rate, however expression of estrus was determined to significantly influence conception rate (<i>P</i>=0.043). Seventy-five LB piglets were weaned across the first three breeding replicates, with parity 2 sows observed to have larger litter sizes than parity 1 sows (<i>P</i>=0.066).</p> <p>Large Black and Duroc-sired (DS) crossbred pigs from replicates 1 and 2 farrowing were fed corn and soybean meal based finishing diets supplemented with (FIB) or without alfalfa and wheat middlings (CON). Following 6 dietary phases through finishing, 25 LB and 25 DS pigs were slaughtered at similar ages for digestive organ dissection and carcass measurements. Loin muscles were evaluated for fresh pork quality and instrumental color and tenderness. LB pigs had a reduced ADG (<i>P</i><0.0001) and G:F (<i>P</i><0.0001) compared to DS pigs. Pigs fed FIB resulted in reduced ADG (<i>P</i>=0.020) and reduced G:F (<i>P</i>=0.007). At slaughter LB pigs were 26.4 kg lighter than DS pigs (<i>P</i><0.0001), and pigs that were fed FIB had lighter live weights (<i>P</i>=0.002) than pigs fed CON. LB pigs had 28.5±1.3 cm² smaller longissimus muscle area (<i>P</i><0.0001), yielding 2.0 cm more 10^th rib back fat than DS pigs (<i>P</i><0.0001). CON pigs had heavier HCW (<i>P</i><0.0001) than FIB pigs, however FIB pigs had greater percent lean (<i>P</i>=0.015). LB pigs had significantly reduced percent lean than DS pigs (<i>P</i><0.0001). LB pigs had loins with reduced drip loss (<i>P</i>=0.009) and cooked shear force values (<i>P</i><0.0001). Overall, the growth and carcass composition of the pigs was most affected by genotype, and to a lesser extent than the type of diet fed. </p> <p>In conclusion, the genetic importation of LB semen was successful as ½ blood piglets were created for dispersal into the U.S. LB herd. Improvements in FTS utilization in this heritage breed contributed to the successful creation of live-born pigs. Additionally, growth and carcass information was obtained for LB breeders to use in understanding and marketing of this heritage breed of pigs.</p>