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Abstract Birth of offspring of a pre-determined sex using flow cytometrically sorted fresh spermatozoa was first achieved in rabbits by Johnson et al. (1989). Since then offspring have been produced using sex-sorted spermatozoa from several different species (reviewed by Johnson, 2000). Initially, efficiency of the sex-sorting technology was poor with only low numbers of spermatozoa sorted per hour. Thus, the offspring derived from flow cytometrically sorted spermatozoa were produced with the use of artificial reproductive technologies (ART) such as in vitro fertilisation (IVF) and culture (IVC), intracytoplasmic sperm injection (ICSI) and deep artificial insemination (AI) which facilitated low dose insemination of potentially compromised spermatozoa. More recently, the development of high-speed sorters (Johnson and Welch, 1999) has facilitated the production of offspring using conventional AI techniques with low dose inseminates (Seidel et al., 1999) and successful cryopreservation of sorted spermatozoa (Schenk et al., 1999; Johnson et al., 2000; Lindsey et al., 2002; Schenk and DeGrofft, 2003). Increased efficiency of sorting bull spermatozoa has evolved through significant instrumentation and biological developments which have enabled the commercialization of the sperm sexing technology in the dairy industry, although conception rates in cows after low dose AI with sexed frozen-thawed spermatozoa are still lower than after standard frozen semen AI (Seidel et al., 1999). Subsequently, over 20 000 calves of pre-determined sex have been produced from commercially available sex-sorted frozen-thawed bull spermatozoa (Seidel, 2003). However, similar developments have not been made in the sheep industry and were examined in this thesis. In this study, successful cryopreservation of sex-sorted ram spermatozoa and production of offspring of the pre-determined sex (X: 94.4 %; Y: 100 %) was achieved after low dose (2-4 x 106 total) insemination using conventional laparoscopic intrauterine (IU) AI. However, the overall pregnancy rate for ewes inseminated with sex-sorted frozen-thawed spermatozoa was low (25 %) compared to ewes inseminated with a commercial dose (140 x 106 total) of non-sorted frozen-thawed spermatozoa (54 %). Cryopreservation has been found to not only reduce the proportion of motile spermatozoa, but cause the remaining spermatozoa to undergo changes that advance membrane maturation thereby shortening their lifespan, especially after in vivo fertilisation (Gillan and Maxwell, 1999). It was found that sorting prior to cryopreservation accelerated the maturation of sperm membranes and after co-incubation with oviducal cells in vitro, sorted frozen-thawed spermatozoa were released more rapidly than non-sorted (control) frozen-thawed spermatozoa. The potentially reduced lifespan of sorted frozen-thawed spermatozoa, and practical constraints on the number of spermatozoa that can be sorted for an insemination dose, makes insemination close to the site of fertilisation and time of ovulation critical for successful fertilisation. After treatment of ewes with GnRH to increase the precision of insemination in respect to the time of ovulation, there was no difference in pregnancy rate between ewes inseminated before, during or after the assumed time of ovulation. Furthermore, there was no difference in pregnancy rate after IU AI with similar doses of sorted frozen-thawed and non-sorted frozen-thawed spermatozoa in GnRH-treated ewes. The minimum dose of sorted frozen-thawed spermatozoa required for commercially acceptable pregnancy rates determined after IU AI was high (20 x 106 motile). Consequently, alternative methods for efficiently producing large numbers of offspring of a pre-determined sex using flow cytometrically sorted ram spermatozoa were investigated. Ram spermatozoa can be stored for short periods of time in a chilled state (liquid storage) or for an indefinite period of time in a frozen state (frozen storage; Salamon and Maxwell, 2000). The fixed location of the sperm sorter requires the need for transport of semen from the point of collection to the site of sorting and processing, but also from the sperm sorter site to the recipient females under artificial conditions. In this study, ram spermatozoa liquid stored for 24 h prior to sorting were efficiently sorted, frozen, thawed and after in vitro fertilisation and culture produced a high proportion of grade 1 blastocysts. Similarly, spermatozoa stored at reduced temperatures after sorting maintained high sperm quality for up to 6 days. Furthermore, frozen-thawed spermatozoa from rams and some non-human primates were successfully prepared for sorting and efficiently sorted producing spermatozoa with high quality in vitro parameters. The quality of frozen-thawed ram spermatozoa after sorting was such that successful re-cryopreservation after sorting was possible. Low numbers of frozen-thawed sorted and re-frozen and thawed spermatozoa were optimal for IVF and a high proportion of grade 1 in vitro embryos of a pre-determined sex were produced. These embryos were either transferred immediately or vitrified prior to transfer, extending the application of the sperm sexing technology further. The birth of lambs of pre-determined sex after transfer of both fresh and vitrified embryos derived from frozen-thawed sorted spermatozoa was achieved. The findings in this thesis suggest that sorted frozen-thawed ram spermatozoa may have more advanced membrane maturation state than non-sorted frozen-thawed spermatozoa, resulting in a decreased fertilizing lifespan in the female reproductive tract. Despite this, the use of sexed ram spermatozoa in a number of physiological states (fresh, liquid, frozen) with several different ARTs is possible in producing significant numbers of offspring of a pre-determined sex. Improved efficiency in both sperm sexing and associated reproductive technologies is required for commercialization to be achieved in the sheep industry.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.

Thesis (M. Sc.) Uppsala : Sveriges lantbruksuniv.

Thesis (M.Nurs.)--University of Hong Kong, 2003. / Includes bibliographical references (leaves 109-118). Also available in print.

Thesis (M.S. in animal science)--Washington State University, May 2007. / Includes bibliographical references.

Made available in DSpace on 2015-03-03T11:52:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-26Bitstream added on 2015-03-03T12:06:08Z : No. of bitstreams: 1 000809964.pdf: 1108463 bytes, checksum: 43fa89dc624ca99b382ecaa200b42518 (MD5) / A utilização da transgenia com a proteína fluorescente verde (GFP) como marcador de células de origem fetal nas placentas de clones bovinos servirá de modelo inédito para estudo morfofisiológico e imunológico da interação materno-fetal, visto que possibilitará o seu mapeamento, diferenciando as células fetais das maternas. Tal modelo terá aplicação direta, principalmente porque estes são animais que apresentam problemas em relação ao seu desenvolvimento. Com o auxílio deste modelo, pretende- se verificar o transporte de substâncias entre a mãe e o feto via endocitose, pela imunolocalização das proteínas chamadas de caveolinas. Para tanto foram utilizados 06 bovinos clonados e 30 bovinos de inseminação artificial (IA) com idade até 90 dias de gestação, os quais tiveram seu desenvolvimento interrompido mediante abate humanitário das receptoras e ovariosalpingohisterectomia, com posterior recuperação do útero gestante. Foram coletados os placentônios e o cório. Uma parte das amostras foi recortada e fixada, por imersão, em solução de parafolmaldeído a 4% ou formoldeído a 10% em tampão fosfato de sódio (PBS) a 0,1M pH 7.4, solução de Zamboni (4% de paraformoldeído, 15% de ácido pícrico, em tampão fosfato de sódio a 0,1M pH 7.4), metacarn (60% de metanol, 30% de clorofórmio, e 10% de ácido acético glacial), para verificação da morfologia e realização de imuno-histoquímica para as proteínas caveolinas -1 e -2 (CAV -1 e CAV-2). As caveolinas -1 foram localizadas nos vilos fetais e maternos, mas sua marcação mais forte foi observada no estroma endometrial. As caveolinas -2 tiveram marcação positiva no trofoblasto e membrana corioalantoide, e, especificamente em célula trofoblástica gigante binucleada. Sendo assim, os resultados mostram que a proteína CAV-1 teve uma maior expressão em relação a proteína CAV-2 e que as proteínas CAV-1 e -2 são parte da composição das cavéolas, sendo . / The transgenic application of green fluorecent protein (GFP) as fetal cell marker on cattle cloned placenta could provide an exclusive model for studying the morphologic and immunologic maternal-fetal interactions, providing information about its mapping, distinguishing the fetal from maternal cells. This model will have direct application, mainly because these are animals that present problems during its development. With this model's support, we intend to verify the substances transport between mother and fetus during endocytocis, through the immunolocalization of protein named caveolae. For these, we used 06 cloned bovine and 30 cattle samples of artificial insemination (AI) with 90 days of pregnancy, which had been their development interrupted by humanitarian slaughter of the recipient and recovery of the pregnant uterus. We collected the placentome and the chorion. A part of the samples will be cutted and fixed, by immersion, on a solution containing 4% of parafomaldehyde or 10% of formaldehyde on a sodium phosphate buffer (PBS) at 0,1M pH 7.4, Zamboni solution (4% of paraformaldehyde, 15% of picric acid, on sodium phosphate buffer 0,1M pH 7.4), metacarn (60% of metanol, 30% of chloroform, and 10% glacial acetic acid), for morphologic and immunohistochemistry verification for caveolinas proteins -1 and -2 ( -1 CAV and CAV- 2). The caveolinas -1 were found in fetal and maternal villi , but its strongest staining was observed in the endometrial stroma . The caveolinas -2 had positive staining in trophoblast and chorioallantoic membrane , and specifically in giant trophoblastic binucleated cell . therefore the results were compared between cloned cattle and from AI or natural mating, for assisting on detection of the reason of many placental alterations, embryonic losses, spontaneous abortion, post-natal mortality and large offspring syndrome on laboratory-manipulated animals. The result suggests that the proteins caveolins -1 and ...

Made available in DSpace on 2016-03-07T19:21:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-12-11. Added 1 bitstream(s) on 2016-03-07T19:25:30Z : No. of bitstreams: 1 000858937.pdf: 237984 bytes, checksum: 61c941e361051e9de4cce1254082d23f (MD5) / O presente estudo avaliou se a concentração de P4 durante o desenvolvimento do folículo ovulatório em protocolo a base de P4/BE e com uma aplicação de GnRH em seu início interfere na prenhez à IATF em vacas Holandesas em lactação. A hipótese é que vacas com maior P4 no dia da aplicação da 1ª dose de PGF2α terão maior fertilidade. Para alterar a P4 os animais (n=594) foram distribuídos aleatoriamente em dois grupos para receberem um ou dois dispositivos intravaginais de P4 (CIDR®, Zoetis, SP, Brasil). O protocolo utilizado foi: no d-11 inserção de 1 CIDR (novo ou previamente usado por 9 dias) + aplicação de 2mg i.m. de BE (Gonadiol®, Zoetis, SP, Brasil) + 100mcg i.m. de GnRH (Cystorelin®, Merial, SP, Brasil); d-4, aplicação de 25mg i.m. de Dinoprost (Lutalyse®, Zoetis, SP, Brasil); d-2, aplicação de 25mg i.m. de Dinoprost + 1mg i.m. de ECP (ECP®, Zoetis, SP, Brasil) + retirada do dispositivo de P4; d0, IATF. Os animais do grupo 2CIDR receberam um CIDR adicional no d-11, o qual foi retirado no d-4. Nos d-11 (n= 117), d-4 (n= 351), d0 (n= 214) e d10 (n= 72) foram colhidas amostras de sangue para determinação da concentração de P4. A P/IA foi determinada através de US nos d32 (DG1) e d60 (DG2). Dados binários foram analisados pelo PROC GLIMMIX, e os dados contínuos pelo PROC MIXED do SAS (foi considerada diferença significativa se P<0,05 e tendência se P<0,1). A P4 não diferiu entre os tratamentos no d-11 (1CIDR= 4,2±0,4 ng/ml; 2CIDR= 4,5± 0,4ng/ml; P>0,1), e no d-4 (1CIDR= 3,5±0,2ng/ml; 2CIDR= 3,8±0,2 ng/ml; P>0,1), porém foi detectada interação entre tratamento e presença de CL no início do protocolo na P4 no d-4 (sem presença de CL e 1CIDR= 2,7±0,3ng/ml; 2CIDR= 3,6±0,3ng/ml, com presença de CL e 1CIDR= 4,3±0,3ng/ml; 2CIDR= 4,0±0,3ng/ml; P<0,05). Não houve diferença na prenhez a IATF e nas perdas gestacionais entre os tratamentos [DG1: 1CIDR= 20,0 ... / The present study evaluated if P4 concentration during ovulatory follicle development in a timed artificial insemination (TAI) protocol based on P4/E2 affects pregnancy per AI in lactating Holstein cows. Our hypothesis is that cows that present higher P4 concentration at PGF2α administration have also higher fertility. To alter P4, animals (n=594) were randomly assigned to receive one or two intravaginals devices of P4 (CIDR®, Zoetis, SP, Brazil). TAI protocol utilized was: d-11 intravaginal device of P4 (new or previously used by 9 days) + 2mg im EB (Gonadiol®, Zoetis, SP, Brazil) + 100mcg im GnRH (Cystorelin®, Merial, SP, Brazil); d-4, 25mg im Dinoprost (Lutalyse®, Zoetis, SP, Brazil); d-2, 25mg im Dinoprost + 1mg im ECP (ECP®, Zoetis, SP, Brazil) + CIDR removal; d0, TAI. Animals in the group 2CIDR received an adicional CIDR at d-11, which was removed at d-4. At d-11 (n=117), d-4 (n=351), d0 (n=214) and d10 (n=72), blood samples were taken from cows for P4 concentration measurements. Pregnancy per AI was determined by ultrasound at d32 (DG1) and d60 (DG2). The binomial data were analyzed using PROC GLIMMIX and continuous data using PROC MIXED of SAS. An effect was considered significant when P<0.05 and tendency when P<0.1. P4 did not differ among treatments at d-11 (1CIDR=4.2±0.4ng/ml; 2CIDR=4.5±0.4ng/ml; P>0.1), and at d-4 (1CIDR=3.5±0.2ng/ml; 2CIDR=3.8±0.2 ng/ml; P>0.1). An interaction was detected between treatment and CL presence at the beginning of TAI protocol in P4 at d-4 (without CL and 1CIDR=2.7±0.3ng/ml; 2CIDR= 3.6±0.3ng/ml, with CL and 1CIDR=4.3±0.3ng/ml; 2CIDR= 4.0±0.3ng/ml; P<0.05). There was no difference among treatments in pregnancy per AI and pregnancy loss between DG1 and DG2 [DG1: 1CIDR= 20,0% (81/310) vs. 2CIDR= 15,7% (65/284); DG2: 1CIDR= 18,2% (74/310) vs. 2CIDR= 13,8% (57/283); Pregnancy loss: 1CIDR= 14,1% (7/81) vs. 2CIDR= 11,5% (7/64); P>0,1]. 37,0% (28/63)...

Made available in DSpace on 2014-06-11T19:25:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-08-11Bitstream added on 2014-06-13T18:53:22Z : No. of bitstreams: 1 pinheiro_vg_me_botib.pdf: 219390 bytes, checksum: a4a81d46d63eddc4df849062b1b592d7 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Existem relatos na literatura que tanto a remoção temporária de bezerro (RTB) como a administração de eCG podem aumentar a eficiência (taxa e prenhez) de tratamentos hormonais com progesterona/progestágenos, em vacas em anestro pós-parto. No presente trabalho a associação de RTB e/ou a aplicação de eCG foram testadas simultaneamente em protocolos hormonais com progesterona, comumente utilizado para inseminação artificial em tempo fixo (IATF) de vacas no período pós-parto. Os protocolos hormonais foram testados em 3 propriedades, em vacas Nelore lactantes (40 a 80d pós-parto, n = 443), com escore de condição corporal de 2,5 a 3,0 (escala de 1 a 5). Em estágio aleatório do ciclo estral (D0), os animais do grupo PEPE (Progesterona-Estrógeno- Prostaglandina-Estrógeno) receberam dispositivo intravaginal contendo progesterona (1,0 g, DIB®, Sintex, Buenos Aires, Argentina) e 2,5 mg de benzoato de estradiol (BE, via IM, Estrogin®, Farmavet, São Paulo, Brazil). Oito dias mais tarde (D8) administrou-se PGF2 (150 μg de d-cloprostenol, via IM, Prolise®, ARSA S.R.L., Buenos Aires, Argentina), e o DIB foi removido. Vinte e quatro horas após a remoção do DIB, as vacas foram tratadas com BE (1,0 mg, IM) e 30 a 36 h mais tarde todos os animais receberam IATF, sem observação do cio. As vacas foram divididas ao acaso em 4 Grupos: PEPE, PEPE/RTB, PEPE/eCG e PEPE/RTB/eCG. No grupo PEPE/RTB, além do tratamento descrito acima (PEPE) os bezerros foram retirados das vacas por 54 – 60 h consecutivas (a partir da retirada do DIB até a IATF). No Grupo PEPE/eCG, os animais receberam o mesmo tratamento do grupo PEPE associado a uma aplicação de eCG (300 UI, via IM, Novormon®, Sintex, Buenos Aires, Argentina) no momento da administração da PGF2 (D8). No Grupo PEPE/RTB/eCG, os animais foram tratados de acordo com o protocolo PEPE/RTB associado a aplicação de eCG (300 UI) no D8... / Reports indicate that either TCR or eCG administration can increase the efficiency (pregnancy rate) of hormonal treatments with progestins during postpartum anestrus. This experiment evaluated effects of TCR and/or eCG administration in a protocol with progesterone that is frequently used for fixed-time artificial insemination (FTAI) in cows during postpartum anestrus. The protocols were tested at three farms in lactating Nelore cows (40 to 80 d postpartum, n = 443) with body condition scores from 2.5 to 3.0 (0- to 5- point scale). At random stage of the estrous cycle (D0), animals received a basic PEPE (Progesterone-Estrogen-Prostaglandin-Estrogen) protocol with insertion of an intravaginal device with progesterone (1.0 g, DIB®, Sintex, Buenos Aires, Argentina) and injection of 2.5 mg of estradiol benzoate (EB, i.m., Estrogin®, Farmavet, São Paulo, Brazil). Eight days later (D8) cows were treated with PGF2; (150 μg d-cloprostenol, i.m., Prolise®, ARSA S.R.L., Buenos Aires, Argentina), and DIB was removed. Twenty four hours after DIB removal, cows received EB (1.0 mg, i.m), and 30 to 36 h later all animals received FTAI, without estrus detection. Cows were allocated randomly to 4 Groups: PEPE, PEPE/TCR, PEPE/eCG and PEPE/TCR/eCG. In Group PEPE/TCR, calves were removed temporarily for 54 – 60 h (from DIB removal until FTAI). In Group PEPE/eCG, animals received PEPE treatment plus one dose of eCG (300 IU, i.m., Novormon®, Sintex, Buenos Aires, Argentina) following PGF2 administration (D8). In Group PEPE/TCR/eCG, animals were treated as in protocol PEPE/TCR plus eCG on D8. All animals were examined by ultrasonography (Aloka SSD 500, 7.5 MHZ probe) 10 days before and at the beginning... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:31:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-17Bitstream added on 2014-06-13T20:21:56Z : No. of bitstreams: 1 alves_ae_dr_jabo.pdf: 382934 bytes, checksum: 26d3285aa222f5838b3d67c55e5d0837 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo deste estudo foi avaliar a taxa de concepção em cadelas após inseminação artificial intrauterina por videolaparoscopia. Um total de 20 cadelas foram inseminadas, sendo 10 com sêmen fresco e 10 com sêmen congelado. Os animais se encontravam em estro natural e sob acompanhamento da citologia vaginal. Quando aproximadamente 90% das células se apresentaram cornificadas, foram realizadas dosagens séricas de progesterona afim de se determinar o momento exato da inseminação. Três machos foram utilizados como doadores de sêmen e as coletas foram realizadas por manipulação digital. Após coleta o ejaculados foram analisados quanto ao volume, motilidade, vigor e concentração espermática. Para a inseminações utilizando sêmen fresco, a coleta foi realizada momentos antes do procedimento, e o sêmen acondicionados em banho–maria a 37°C até o momento da inseminação. Amostras destinadas a inseminação com sêmen descongelado, obtiveram mesmo processo de análises após coleta, seguindo para o processo de congelação, permanecendo congeladas por no mínimo uma semana, e então descongeladas momentos antes do procedimento. De acordo com os concentrações séricas de progesterona, as inseminações intrauterinas por videolaparoscopia foram realizadas, sendo cada corno uterino inseminado com 1mL de sêmen, em concentração de 250x106 espermatozóides/mL nas cadelas inseminadas com sêmen fresco e 80x106 espermatozóides/mL naquelas inseminadas com sêmen congelado. Após 7 dias as cadelas foram ováriosalpingohisterectomizadas, sendo o lúmen das tubas e cornos uterinos lavados com solução de PBS. Embriões foram encontrados nos lavados de 7 cadelas inseminadas com sêmen fresco, e em 5 inseminadas com sêmen descongelado. Concluiu-se que o procedimento de inseminação artificial intrauterina... / The objective of this study was to evaluate the fertility in bitches after intrauterine artificial insemination by videolaparoscopy. A total of 20 bitches were inseminated, being 10 with fresh semen and 10 with frozen. The animals were in natural oestrus, and their vaginal cytology was followed. When approximately 90% of the cells were cornificated, evaluation of serum levels of progesterone was performed, with the objective to determinate the exact moment of the artificial insemination. Three males were used as donors, and the samples collection was performed by digital manipulation. After collection the ejaculates were analysed considering volume, motility, vigor and sperm concentration. For the inseminations using fresh semen the collection was performed minutes before the process, and the semen sample was maintained at 37°C. Samples destined to inseminations using frozen semen were submitted to the same analysis process after collection, followed by the freezing process, being frozen during a minimum period of one week, and thawing moments before the procedure. According to the serum levels of progesterone, the intrauterine inseminations by videolaparoscopy were performed, being each uterine corn inseminated with 1 mL of semen, with 250x106 espermatozoa/mL in bitches with fresh semen and 80x106 espermatozoa/mL in bitches with frozen semen. After 7 days the bitches were ovariohysterectomized, and the lumen of the uterine tube and corns were flushed with PBS solution. Embryos were found in 7 bitches inseminated with fresh semen, and in 5 inseminated with frozen semen. It was concluded that the intrauterine artificial insemination by videolaparoscopy seems to be an interesting reproductive biotechnology method, especially in what refers to the use of frozen semen.