A técnica de inseminação artificial (IA) por laparoscopia em ovinos foi utilizada pela primeira vez em 1982, o que tornou viável a utilização econômica do sêmen congelado nesta espécie. Os experimentos foram realizados com o objetivo de determinar as taxas de prenhez de ovelhas inseminadas por laparoscopia, utilizando 50 x 106 espermatozóides descongelados e capacitados in vitro. No experimento 1, foi utilizado sêmen de um reprodutor e 67 ovelhas com estros sincronizados, divididas aleatoriamente entre os grupos: sêmen congelado (SC1) e sêmen congelado capacitado in vitro (SCC1). As ovelhas foram submetidas à IA, com 50x106 espermatozóides, 56 e 60 horas, respectivamente, após a retirada das esponjas impregnadas com progesterona. No grupo SCC1, a taxa de prenhez foi de 48,39% (15/31) significativamente superior a do grupo SC1 de 25% (9/36). No experimento 2, utilizou-se um reprodutor e 100 ovelhas com os estros sincronizados, distribuídas aleatoriamente entre três grupos: sêmen fresco (SF2), SC2 e SCC2. Nos grupos SF2 e SC2 as fêmeas foram inseminadas com 100x106 espermatozóides e 56 horas após a retirada da progesterona. No grupo das fêmeas inseminadas com espermatozóides capacitados in vitro, utilizou-se 50x106 espermatozóides e um intervalo de 60 horas após retirada da progesterona. As taxas de prenhez diagnosticadas nos diferentes grupos experimentais foram: SF2 60,9% (25/41), SC2 56,70% (17/30) e SCC2 44,90% (13/29), não existindo diferença estatística entre os grupos. Os experimentos realizados mostraram a viabilidade doemprego, na espécie ovina, da IA por laparoscopia utilizando 50x106 espermatozóides descongelados e capacitados in vitro. / The ovine artificial insemination (AI) by laparoscopy was described the first time in 1982, since this time frozen semen became a tool for use under field conditions. Today one research goal is to achieve high preganancies rates after AI using a reduced number of frozen/thawed spermatozoa per dosis. The experiments were carried out with the objective to determine the pregnancy rate of ewes inseminated by laparoscopy, using 50 x 106 thawed and in vitro capacited spermatozoa. In the experiment 1 was used semen of one fertily ram and 67 ewes with synchronized estrus. The females were randomly distributed among two experimental groups: inseminated with frozen semen (SC1) or frozen and in vitro capacitated semen (SCC1). The ewes were submitted to the AI with 50x106 spermatozoa, 56 (SC1) or 60 (SCC1) hours, respectively, after retreat of the intra vaginal device impregnated with progesterona. In the group SCC1 the pregnancy rate was 48,39% (15/31) significantly different from the 25% (9/36) observed in the group SC1. In the experiment 2 was used one fertily ram and 100 ewes with synchronized estrus, distributed randomly among three groups: inseminated with fresh semen (SF2), 100 x 106 spermatozoa, SC2, 100 x 106 frozen spermatozoa and SCC2, 50 x 106 frozen and in vitro capacited spermatozoa. The moment of the laparoscopic AI was the same as used in the experiment 1: 56 (SF2 and SC2) or 60 (SCC2) hours, respectively, after retreat of the intra vaginal device impregnated with progesterona. The observed pregnancy rates were similar among the experimental groups: SF2 60,9% (25/41), SC2 56,70% (17/30) and SCC2 44,90% (13/29). Our experiments showed the ability of frozen/thawed and in vitro capacitated ovine spermatozoa to in vivo fertilize and promote the development of pregancies. In conclusion, itis possible to use 50x106 frozen/thawed and in vitro capacited spermatozoa for ovine laparoscopic AI.

Resumo: Algumas avaliações da capacidade de fecundação como a produção in vitro de embriões (PIV) e a coloração vital indicam que a sexagem de espermatozóides por centrifugação em gradiente de densidade pode causar diminuição nas taxas de desenvolvimento embrionário, entretanto, não são conclusivas quanto à avaliação da integridade das membranas espermáticas. Com o intuito de obter dados mais precisos em relação à viabilidade espermática pós-sexagem, foram descongeladas doses de sêmen de 6 touros diferentes, e avaliadas quanto aos danos espermáticos causados pela centrifugação em gradiente descontínuo de Percoll (com densidade variando de 1,111g/mL a 1,123g/mL). Antes e após a centrifugação dos espermatozóides, além das avaliações de concentração, motilidade e morfologia, o sêmen foi ainda avaliado quanto à integridade das membranas plasmáticas, acrossomal e mitocondrial pela associação das sondas fluorescentes: Iodeto de Propídio (PI), aglutinina de Pisum Sativum conjugado a Isotiocianato de flurosceína (FITC-PSA) e Iodeto de 5,5',6,6'-tetracloro- 1,1,3,3'-tetraetilbenzimidazolilcarbocianina (JC-1). Os resultados demonstraram que o processo de separação de espermatozóides X e Y por centrifugação em gradiente de densidade promoveu uma melhora nas características associadas à motilidade progressiva, além de uma significativa diminuição na incidência de defeitos maiores. Ainda nas amostras pós-centrifugação, foi observado um aumento no percentual de células com Membrana Plasmática Intacta e com Alto Potencial Mitocondrial. No entanto, o percentual de células com Membrana Acrossomal Intacta foi sensivelmente diminuída provavelmente devido ao fenômeno de reação acrossomal desencadeado pela centrifugação. / Abstract: Some fecundity evaluations as the in vitro embryo production (IVP) and the vital stains have indicating that the density gradients centrifugation techniques can reduce the embryo development rates. However they are not conclusive for the integrity of spermatic membranes. Wit the purpose of obtain more precise sperm viability data after the sexing procedure, frozen semen samples from six different bulls were thawed and assessed for spermatic damage caused by centrifugation in discontinuous Percoll gradient (with density varying from 1,111g/mL to 1,123g/mL). Before and after the separation of X-bearing bovine sperm, were evaluated the sperm concentration, motility and morphology. Moreover the semen samples were assessed for integrity of plasma, acrosome and mitochondrial membranes by fluorescent probes association: propidium iodide (PI), fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). The results demonstrated that the X-bearing sperm separation process by density gradient centrifugation caused an increase in the progressive motility parameters and a decrease in the major defects incidence. In addition, it was observed an increase in the cells categories with Intact Plasma Membrane and High Mitochondrial Membrane potential in the post-centrifugation samples. Nevertheless, the percentage of cells with Intact Acrossome Membrane was reduced probably due to the acrosome reaction phenomenon induced by centrifugation. / Orientadora: Vera Fernanda Martins Hossepian de Lima / Coorientador: Rubens Paes de Arruda / Banca: Gisele Zoccal Mingoti / Banca: Eneiva Carla Carvalho Celeghini / Mestre

A água empregada na produção de doses inseminantes (DIs) de suínos pode ser um dos veículos de introdução de bactérias contaminantes, podendo levar à perda de qualidade das mesmas. O objetivo do presente estudo foi avaliar o número de mesófilos aeróbios totais (MAT) na água utilizada em centrais de inseminação de suínos e sua influência nas contagens bacterianas das DIs produzidas. Foram conduzidos três ciclos de amostragem em seis centrais de inseminação (A-F), intencionalmente incluídas no estudo de acordo com os critérios: estar localizada no sul do Brasil; produzir >1.000 DIs mensais; e concordar em participar do estudo. A cada ciclo, eram colhidas amostras de água antes da purificação, água após a passagem pelo sistema de purificação, água após armazenamento, diluente, sêmen in natura de três machos distintos de fertilidade comprovada; e das respectivas doses inseminantes preparadas com sêmen e diluente amostrados. A água colhida antes do sistema de purificação foi avaliada quanto ao número mais provável (NMP) de coliformes totais e Escherichia coli, em 100 mL. pela técnica dos tubos múltiplos. Todas as amostras colhidas foram avaliadas quanto ao número de mesófilos aeróbios totais (MAT), por meio da semeadura em profundidade em Ágar para Contagem (PCA). Todas as 18 amostras de água colhidas antes da purificação apresentaram <1,1 NMP.100 mL-1 de Escherichia coli. A média de MAT nas amostras de água variou entre 0,1 log UFC.mL-1 na água após-purificação da central E e 3,5 log.UFC.mL-1 na água antes da purificação da central F. Não houve diferença significativa (P>0,05) entre as contagens médias de MAT obtidas em diferentes tipos de amostra de água e entre as centrais de inseminação. A média de MAT nas amostras de diluente variou de 0,16 log UFC. mL-1 à 2,78 log UFC.mL-1. As centrais D e F apresentaram diluentes significativamente (P<0,05) mais contaminados. No sêmen, a média de MAT variou de 1,75 log. UFC.mL-1 à 3,79 log UFC.mL-1 e nas DIs entre 0,73 log.UFC.mL-1 e 2,88 log UFC.mL-1. A central de inseminação F apresentou média de MAT significativamente (P<0,05) maior que as demais centrais. Considerando os padrões existentes, conclui-se que a água captada, purificada e armazenada apresentou boa qualidade microbiológica em todas as centrais de inseminação e não influenciou a média de mesófilos aeróbios totais das DIs preparadas. O incremento de bactérias, quando observado, foi provavelmente resultante da contaminação de origem ambiental durante o preparo do diluente e das doses inseminantes. / The water employed in the production of insemination doses (IDs) of swine may be one of the vehicles for introduction of contaminant bacteria, and can lead to their quality loss. The objective of this study was to assess the number of total aerobic mesophilic (TAM) in water used in swine artificial insemination centers (SAIC) and its influence in the total bacterial counting of the IDs produced. Three cycles of sampling were conducted in six SAIC (A-F) intentionally included in the study according to the following criteria: to be located in the south of Brazil, to produce > 1.000 (IDs) monthly, and to agree in participating of the study. At each cycle, samples were collected from water before the purification, water after passage through the purification system, water after storage, extenders, fresh semen collected from three different boars and the respective (ID) prepared with the semen and extender sampled. The water collected before the purification system was evaluated for total coliforms and Escherichia coli most probable number (NMP) in 100 mL using the multiple tubes technic. All samples collected were evaluated for the number of total aerobic mesophilic (TAM) by the pour plate technic. All the 18 samples of water collected before the purification presented < 1.1 NMP. 100 mL-1 of Escherichia coli. The average of TAM in the water samples varied between 0.1 log.CFU. ml-1 in the water after purification of the central E and 3.5 log. CFU.mL-1 in the water before purification of the central F. There was no significant difference (P>0.05) among the average counting of TAM obtained in different water samples types and among SAICs. The average TAM in samples of diluents varied from 0.16 log.CFU.mL-1 to 2.78 log.CFU.mL-1; the SAICs D and F presented diluents significantly (P< 0.05) more contaminated. Among the semen samples, the TAM average varied from 1.75 log.CFU.mL-1 to 3.79 log CFU.mL-1 and in the DIs between 0.73 log.CFU.mL-1 and 2.88 log.CFU.mL-1. The SAIC F presented average of TAM significantly (P<0.05) higher than the others SAICs. Considering the available standards, it was concluded that water collected, purified and stored presented a good microbiological quality in all insemination centers and did not influence the number of TAM in the prepared IDs. The increase on bacterial population may have resulted from contamination of environmental origin during the preparation of the extender and insemination doses.

A técnica de inseminação artificial (IA) por laparoscopia em ovinos foi utilizada pela primeira vez em 1982, o que tornou viável a utilização econômica do sêmen congelado nesta espécie. Os experimentos foram realizados com o objetivo de determinar as taxas de prenhez de ovelhas inseminadas por laparoscopia, utilizando 50 x 106 espermatozóides descongelados e capacitados in vitro. No experimento 1, foi utilizado sêmen de um reprodutor e 67 ovelhas com estros sincronizados, divididas aleatoriamente entre os grupos: sêmen congelado (SC1) e sêmen congelado capacitado in vitro (SCC1). As ovelhas foram submetidas à IA, com 50x106 espermatozóides, 56 e 60 horas, respectivamente, após a retirada das esponjas impregnadas com progesterona. No grupo SCC1, a taxa de prenhez foi de 48,39% (15/31) significativamente superior a do grupo SC1 de 25% (9/36). No experimento 2, utilizou-se um reprodutor e 100 ovelhas com os estros sincronizados, distribuídas aleatoriamente entre três grupos: sêmen fresco (SF2), SC2 e SCC2. Nos grupos SF2 e SC2 as fêmeas foram inseminadas com 100x106 espermatozóides e 56 horas após a retirada da progesterona. No grupo das fêmeas inseminadas com espermatozóides capacitados in vitro, utilizou-se 50x106 espermatozóides e um intervalo de 60 horas após retirada da progesterona. As taxas de prenhez diagnosticadas nos diferentes grupos experimentais foram: SF2 60,9% (25/41), SC2 56,70% (17/30) e SCC2 44,90% (13/29), não existindo diferença estatística entre os grupos. Os experimentos realizados mostraram a viabilidade doemprego, na espécie ovina, da IA por laparoscopia utilizando 50x106 espermatozóides descongelados e capacitados in vitro. / The ovine artificial insemination (AI) by laparoscopy was described the first time in 1982, since this time frozen semen became a tool for use under field conditions. Today one research goal is to achieve high preganancies rates after AI using a reduced number of frozen/thawed spermatozoa per dosis. The experiments were carried out with the objective to determine the pregnancy rate of ewes inseminated by laparoscopy, using 50 x 106 thawed and in vitro capacited spermatozoa. In the experiment 1 was used semen of one fertily ram and 67 ewes with synchronized estrus. The females were randomly distributed among two experimental groups: inseminated with frozen semen (SC1) or frozen and in vitro capacitated semen (SCC1). The ewes were submitted to the AI with 50x106 spermatozoa, 56 (SC1) or 60 (SCC1) hours, respectively, after retreat of the intra vaginal device impregnated with progesterona. In the group SCC1 the pregnancy rate was 48,39% (15/31) significantly different from the 25% (9/36) observed in the group SC1. In the experiment 2 was used one fertily ram and 100 ewes with synchronized estrus, distributed randomly among three groups: inseminated with fresh semen (SF2), 100 x 106 spermatozoa, SC2, 100 x 106 frozen spermatozoa and SCC2, 50 x 106 frozen and in vitro capacited spermatozoa. The moment of the laparoscopic AI was the same as used in the experiment 1: 56 (SF2 and SC2) or 60 (SCC2) hours, respectively, after retreat of the intra vaginal device impregnated with progesterona. The observed pregnancy rates were similar among the experimental groups: SF2 60,9% (25/41), SC2 56,70% (17/30) and SCC2 44,90% (13/29). Our experiments showed the ability of frozen/thawed and in vitro capacitated ovine spermatozoa to in vivo fertilize and promote the development of pregancies. In conclusion, itis possible to use 50x106 frozen/thawed and in vitro capacited spermatozoa for ovine laparoscopic AI.

A água empregada na produção de doses inseminantes (DIs) de suínos pode ser um dos veículos de introdução de bactérias contaminantes, podendo levar à perda de qualidade das mesmas. O objetivo do presente estudo foi avaliar o número de mesófilos aeróbios totais (MAT) na água utilizada em centrais de inseminação de suínos e sua influência nas contagens bacterianas das DIs produzidas. Foram conduzidos três ciclos de amostragem em seis centrais de inseminação (A-F), intencionalmente incluídas no estudo de acordo com os critérios: estar localizada no sul do Brasil; produzir >1.000 DIs mensais; e concordar em participar do estudo. A cada ciclo, eram colhidas amostras de água antes da purificação, água após a passagem pelo sistema de purificação, água após armazenamento, diluente, sêmen in natura de três machos distintos de fertilidade comprovada; e das respectivas doses inseminantes preparadas com sêmen e diluente amostrados. A água colhida antes do sistema de purificação foi avaliada quanto ao número mais provável (NMP) de coliformes totais e Escherichia coli, em 100 mL. pela técnica dos tubos múltiplos. Todas as amostras colhidas foram avaliadas quanto ao número de mesófilos aeróbios totais (MAT), por meio da semeadura em profundidade em Ágar para Contagem (PCA). Todas as 18 amostras de água colhidas antes da purificação apresentaram <1,1 NMP.100 mL-1 de Escherichia coli. A média de MAT nas amostras de água variou entre 0,1 log UFC.mL-1 na água após-purificação da central E e 3,5 log.UFC.mL-1 na água antes da purificação da central F. Não houve diferença significativa (P>0,05) entre as contagens médias de MAT obtidas em diferentes tipos de amostra de água e entre as centrais de inseminação. A média de MAT nas amostras de diluente variou de 0,16 log UFC. mL-1 à 2,78 log UFC.mL-1. As centrais D e F apresentaram diluentes significativamente (P<0,05) mais contaminados. No sêmen, a média de MAT variou de 1,75 log. UFC.mL-1 à 3,79 log UFC.mL-1 e nas DIs entre 0,73 log.UFC.mL-1 e 2,88 log UFC.mL-1. A central de inseminação F apresentou média de MAT significativamente (P<0,05) maior que as demais centrais. Considerando os padrões existentes, conclui-se que a água captada, purificada e armazenada apresentou boa qualidade microbiológica em todas as centrais de inseminação e não influenciou a média de mesófilos aeróbios totais das DIs preparadas. O incremento de bactérias, quando observado, foi provavelmente resultante da contaminação de origem ambiental durante o preparo do diluente e das doses inseminantes. / The water employed in the production of insemination doses (IDs) of swine may be one of the vehicles for introduction of contaminant bacteria, and can lead to their quality loss. The objective of this study was to assess the number of total aerobic mesophilic (TAM) in water used in swine artificial insemination centers (SAIC) and its influence in the total bacterial counting of the IDs produced. Three cycles of sampling were conducted in six SAIC (A-F) intentionally included in the study according to the following criteria: to be located in the south of Brazil, to produce > 1.000 (IDs) monthly, and to agree in participating of the study. At each cycle, samples were collected from water before the purification, water after passage through the purification system, water after storage, extenders, fresh semen collected from three different boars and the respective (ID) prepared with the semen and extender sampled. The water collected before the purification system was evaluated for total coliforms and Escherichia coli most probable number (NMP) in 100 mL using the multiple tubes technic. All samples collected were evaluated for the number of total aerobic mesophilic (TAM) by the pour plate technic. All the 18 samples of water collected before the purification presented < 1.1 NMP. 100 mL-1 of Escherichia coli. The average of TAM in the water samples varied between 0.1 log.CFU. ml-1 in the water after purification of the central E and 3.5 log. CFU.mL-1 in the water before purification of the central F. There was no significant difference (P>0.05) among the average counting of TAM obtained in different water samples types and among SAICs. The average TAM in samples of diluents varied from 0.16 log.CFU.mL-1 to 2.78 log.CFU.mL-1; the SAICs D and F presented diluents significantly (P< 0.05) more contaminated. Among the semen samples, the TAM average varied from 1.75 log.CFU.mL-1 to 3.79 log CFU.mL-1 and in the DIs between 0.73 log.CFU.mL-1 and 2.88 log.CFU.mL-1. The SAIC F presented average of TAM significantly (P<0.05) higher than the others SAICs. Considering the available standards, it was concluded that water collected, purified and stored presented a good microbiological quality in all insemination centers and did not influence the number of TAM in the prepared IDs. The increase on bacterial population may have resulted from contamination of environmental origin during the preparation of the extender and insemination doses.

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.

Several methods are used for studying seed dispersal (seed traps, tracking individual seeds, tracking seeds coloured by fluorescent colours, etc.) However, only a few studies compared results obtained by several methods. In first part of this master thesis, I compared the three above mentioned methods used for studying seed dispersal using species from Asteraceae family. From previous research within the study area (Úštěcko), it is known that dry grassland species are able to colonize abandoned fields. Using seed dispersal data obtained within this theses, I tried to assess the role of seed dispersal on distribution of dry grassland species on abandoned fields both on local and regional scale. The results showed that dispersal curves obtained by the three methods differ significantly. This results may be due to different wind conditions during the experiments. Seed trap data show results from long-term seed dispersal influenced by highly variable wind conditions. In contrary, seed release experiments showed results based on single dispersal event under limited wind conditions. Tracking seeds coloured by fluorescent colours was shown to be not convenient for small seeds, but I can recommend this method for larger seeds. Influence of seed dispersal ability on abundance of dry grassland species on...

Thesis (M. Tech.) -- Central University of Technology, Free State, 2007 / Four different South African indigenous (Naked Neck (NN), Ovambo (OVB), Venda (VD) and Potchefstroom Koekoek (PK) chicken breeds were used in this study. From each of the four breeds of chicken, 40 hens and 8 cocks were selected randomly. Two groups each of sixteen cocks were subsequently formed: high performing (HP) and low performing (LP) groups to determine the relationships between cock semen viability and the fertility of artificially inseminated South African indigenous layer breeds. Semen was collected following five minutes of sexual massage (5SM) and evaluated for semen volume (ml), sperm motility (%), live sperm (%) and total sperm (x109/ml). Semen from each cock was then used to inseminate five hens per breed, in each treatment. Each hen was inseminated twice a week throughout the duration of the trial. During the experimental period, each hen was inseminated with 0.05 ml diluted semen. The artificially inseminated hens were examined for average egg weight (g), fertility (%), hatchability of set eggs (%), live chicks (%), normal chicks (%) and chick weight (g). A total of 1600 eggs, i.e. 400 eggs from each breed were collected in three batches following artificial insemination from individually caged hens and were hatched to compare hatching parameters among breeds. The hatchability traits of hens of the four breeds (NN, OVB, PK and VD) were compared. Hatching egg weight had significant (P < 0.05) difference among the four breeds. The results of this study indicate that semen viability exemplified by ejaculate volume, sperm motility; live sperm and total sperm per ejaculate were significantly (P < 0.01) superior in the HP cocks compared to the LP cocks. Hens inseminated with semen from the HP cocks in each experimental group resulted in higher egg weight (g), fertility (%), hatchability of set eggs (%), live chicks (%), normal chicks (%) and chick weight (g). Significant positive relationships existed between semen volume and sperm motility (P < 0.05), semen volume and live sperm cells (P < 0.01), semen volume and total sperm (P < 0.01) in NN, OVB and VD, with negative correlations in PK. Some positive correlations were found between sperm motility and live spermatozoa (P < 0.01), sperm motility and total sperm (P < 0.01), live sperm and total sperm (P< 0.01) in NN, OVB, PK and VND. Fertility was the highest in the HP group. Fertility was also the highest in PK, intermediate and similar in OVB and NN and lowest in VD (P<0.05). Breed had a significant effect on hatchability of fertile eggs (P<0.05). Hatchability of total eggs set was highest in PK and NN, intermediate in OVB and lowest in VD (P<0.05). Breed had a significant effect on live, normal chicks and chick weight (P<0.05). Live chick was the highest in NN, whereas at day-old, normal chick and chick weight at hatching were the highest (23.50 ± 0.11) (P<0.05) in PK (98.14 ± 0.67 vs. 37.90 ± 0.28 g), intermediate and similar in NN (87.90 ± 0.63 vs. 23.50 ± 0.11) and OVB (87.75 ± 0.45 vs. 32.81 ± 0.49 g) and the lowest but with an acceptable value in VD (76.85 ± 0.46 vs. 26.90 ± 0.36 g). There were some correlations among different hatchability traits depending on breed. The correlations were more profound among PK. It was clear that chick weight as percent of egg weight was not just a function of egg weight, and that genotype also played an important role favouring the heavier breeds. The results obtained in this study on the relationships between cock semen viability and the fertility of artificially inseminated South African indigenous layer breeds elucidate that the use of high performing (HP) cocks following five minutes of sexual massage, prior to semen collection and artificial insemination of layers is a practical method for optimising sperm viability and subsequent fertility of hens. The results of this study suggest that the Potchefstroom Koekoek (PK) cocks and hens are superior to the Naked Necks (NN), Ovambo (OVB) and Venda (VD). The Ovambo and Naked Neck cocks ranked second in donating quality semen as well as in improving the fertility and hatchability traits of the indigenous chicken breeds. Thus selection of high performing cocks through five minutes sexual massage prior to semen collection and use is recommended for poultry AI breeding programmes.

Thesis (MScMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Male factor infertility is a general term that describes couples in which an inability to conceive is associated with a problem identified in the male partner. Intrauterine insemination (IUI) together with ovulation induction has been shown to be an effective treatment method for male factor infertility. Oocyte production by the ovaries is stimulated by the use of fertility drugs. A prepared sperm sample is then injected into the uterus through the vagina using an IUI catheter which brings the oocytes and spermatozoa into close proximity. Semen preparation is an integral part of an IUI cycle. In a developing country, a simple inexpensive semen preparation method for IUI procedures, not necessitating a lot of equipment, is essential. An example of such a method, the Sep-D Kit (Surelife Sep-D Kit, Surelife Media Technologies Pty Ltd, Singapore) has been proposed as a possible preparation method. In a pilot study performed by the principal investigator (Roxanne Gentis), comparing the Sep-D Kit and standard swim-up preparation methods, it was found that the Sep-D Kit compared very well with the swim-up method regarding most pre- and post-preparation semen parameters. The Sep-D Kit method, however, still needed further testing to see whether or not pregnancy rates resulting from the method are comparable with that resulting from the standard swim-up method, as this ultimately is the required result of an IUI. The primary aim of this study was to compare the Sep-D Kit method to the standard swim-up method with regards to biochemical pregnancy outcome, post-preparation sperm count, motility, total motile count (TMC), morphology, DNA compaction and fragmentation (CMA3 and TUNEL). The secondary aim was to evaluate which variables, male and female, affect biochemical pregnancy outcome. The study took place at Drs Aevitas Fertility Clinic, Vincent Pallotti Hospital, Pinelands. The study was a prospective analytical study and was conducted from December 2010 until October 2012. A total of 473 IUI cycles were evaluated. Results showed that the Sep-D Kit semen preparation method was non-inferior to the standard swim-up method with regards to biochemical pregnancy rates, post-preparation count and TMC. The swim-up method produced samples with a significantly higher post-preparation motility compared to the Sep-D Kit method, however both methods still managed to produce similar biochemical pregnancy rates (10.39% for the swim-up group versus 11.57% for Sep-D Kit group). For the total cohort of cycles analysed the only female parameter which significantly predicted biochemical pregnancy outcome in this study was age. Sperm motility (post-preparation) was the only male parameter that significantly affected biochemical pregnancy outcome. The Sep-D Kit method is more cost effective and also time saving compared to the swim-up method. There is also no need for expensive laboratory equipment or a trained embryologist using the Sep-D Kit preparation method. The Sep-D Kit may therefore be used with confidence as a standard semen preparation method, and may be implemented in developing countries for use in routine IUI procedures. / AFRIKAANSE OPSOMMING: Manlike faktor infertiliteit is 'n algemene term wat gebruik word om paartjies te beskryf wat 'n onvermoë toon om swanger te raak as gevolg van 'n probleem wat geassosieer word met die man. Die kombinasie van intra-uteriene inseminasie (IUI) en ovulasie induksie kan doeltreffend gebruik word om manlike faktor infertiliteit te behandel. Vrugbaarheidsmiddels word gebruik om oösietproduksie in die die eierstokke te stimuleer en 'n voorbereide spermmonster word dan transvaginaal in die baarmoeder ingespuit om sodoende die spermatozoa en oösiete na-aan mekaar te bring. Semenvoorbereiding is 'n integrale deel van 'n IUI siklus en in 'n ontwikkelende land is 'n eenvoudige, goedkoop semenvoorbereidingsmetode – wat die gebruik van duur toerusting uitsluit – noodsaaklik. Die Sep-D Kit metode (Surelife Sep-D Kit, Surelife Media Technologies Pty Ltd, Singapore) is 'n voorbeeld van so 'n voorbereidingsmetode. 'n Loodsstudie, uitgevoer deur die hoofnavorser, (Roxanne Gentis), het gewys dat die Sep-D Kit en standaard opswem voorbereidingmetodes goed vergelyk ten opsigte van meeste semenparameters voor- en na voorbereiding. Dit is egter ook noodsaaklikheid vir verdere navorsing om vas te stel of swangerskapuitkoms na die gebruik van die twee semenvoorbereidingsmetodess vergelykbaar is, aangesien dit die uiteindelike, verlangde uitkoms van 'n IUI is. Die primêre doel van hierdie studie was om die Sep-D Kit metode te vergelyk met die standaard opswemmetode met betrekking tot biochemiese swangerskapuitkoms asook spermtelling, motiliteit, totale motiele spermtelling (TMS), morfologie, DNA kompaksie en fragmentering (CMA3 en TUNEL) na spermvoorbereiding. Die sekondêre doel was om te evalueer watter veranderlikes, manlik en vroulik, die bichemiese swangerskapuitkoms beïnvloed. Die studie is uitgevoer by die Drs Aevitas Fertiliteitskliniek, Vincent Pallotti Hospitaal, Pinelands. Die studie was prospektief analities en het gestrek vanaf Desember 2010 tot en met Oktober 2012. 'n Totaal van 473 IUI siklusse is evalueer en ontleed. Die resultate van die studie het getoon dat die Sep-D Kit semenvoorbereidingsmetode nie ondergeskik aan die opswemmetode was ten opsigte van biochemiese swangerskap, spermtelling en TMS na semenvoorbereiding nie, Spermmotiliteit was betekenisvol hoër vir die opswemmetode vergelykend met die Sep-D Kit, maar ten spite van die verskil was die biochemiese swangerskapsyfers in die twee groepe nie verskillend nie (10.39% in die opswem groep en 11.57% in Sep-D Kit groep). In die totale kohort siklusse wat ontleed is was dit net die ouderdom van die vrou wat 'n betekenisvolle effek op biochemiese swangerskapuitkoms gehad het. Die enigste manlike faktor wat 'n betekenisvolle effek op biochemiese swangerskapuitkoms gehad het was die motiliteit na semenvoorbereiding. Die Sep-D Kit metode is meer koste-effektief en tydbesparend as die standard opswemmetode. Die uitvoer van die Sep-D Kit metode vereis ook ook geen duur apparaat of 'n opgeleide embrioloog nie. Die Sep-D Kit metode kan dus met vertroue gebruik word as 'n standaard semenvoorbereidingsmetode en kan in ontwikkelende lande vir gebruik tydens roetine IUI prosedures geïmplementeer word.

Thesis (MScMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: CHAPTER 1 A review of the application of traditional semen parameters for the investigation and diagnosis of male infertility and the role of predictive values in assisted reproductive techniques (ART) is presented. The importance of sperm morphology, with special emphasis on sperm morphology evaluation, is discussed. Also presented is an overview of the physiology of sperm DNA, the process of spermatogenesis, as well as the contribution of the spermatozoon to the embryo. The different causes of sperm DNA damage and techniques to determine DNA damage in spermatozoa are described. A survey is presented of the correlation of sperm DNA with sperm morphology. CHAPTER 2 All the materials and methods applicable to this study are described. Sperm morphology assessment and two different sperm DNA tests, the chromomycin A3 (CMA3) staining test and the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) assay, are discussed in detail. CHAPTER 3 Results obtained in this study are presented. Results include the prevalence of abnormal sperm DNA and association with sperm morphology, specifically in the p-pattern and g-pattern morphology groups. Further results include the correlation of sperm morphology and sperm DNA with fertilization in vitro, embryo quality and pregnancy outcome. The percentage CMA3 positive spermatozoa (abnormal DNA) and percentage TUNEL positive spermatozoa (abnormal DNA) had a significant negative association with normal sperm morphology. P-pattern and g-pattern morphology groups differed significantly from each other for both CMA3 and TUNEL. A significant positive association between CMA3 and TUNEL was observed. No association between the percentage normal sperm morphology, percentage CMA3 positive spermatozoa and percentage TUNEL positive spermatozoa and IUI pregnancy outcome was observed. A significant negative association between the percentage TUNEL positive spermatozoa and IVF/ICSI pregnancy outcome was established. The percentage CMA3 positive spermatozoa had a significant positive (unexpected) association with IVF/ICSI pregnancy outcome. There was no association between the three variables and IVF/ICSI fertilization rates. A significant positive association between the percentage normal sperm morphology and IVF/ICSI embryo quality was found. There was a significant positive association between the percentage CMA3 positive spermatozoa and IVF/ICSI embryo quality (unexpected). The percentage TUNEL positive spermatozoa and IVF/ICSI embryo quality was negatively associated. CHAPTER 4 Interpretation of the results and future perspectives are discussed. The CMA3 staining test and TUNEL assay has a limited ability to distinguish between the p-pattern and g-pattern morphology groups. P-pattern spermatozoa are more likely to possess poor chromatin packaging and show increased levels of DNA fragmentation, but some p-pattern patients also may have normal DNA and g-pattern patients abnormal DNA. It is recommended that a sperm DNA test should be implemented routinely in andrology laboratories for the clinical diagnosis of sperm DNA damage in patients. / AFRIKAANSE OPSOMMING: HOOFSTUK 1 'n Samevatting wat handel oor die toepassing van tradisionele semen parameters vir die evaluasie en diagnose van manlike infertiliteit, asook die rol van voorspellingswaardes in kunsmatige voortplantingstegnieke word voorgelê. Die belangrikheid van sperm morfologie, met die klem op sperm morfologie evaluering, word ook bespreek. 'n Oorsig van sperm DNS fisiologie, die proses van spermatogenese, sowel as die sperm se bydrae tot die embrio word hier aangebied. Die verskillende oorsake van sperm DNS skade en die tegnieke om sperm DNS skade vas te stel, asook die die korrelasie tussen sperm DNS en sperm morfologie word ook bespreek. HOOFSTUK 2 Alle materiale en metodes wat van toepassing is op hierdie studie word beskryf. Sperm morfologie evaluering en twee verskillende sperm DNS toetse, die chromomycin A3 (CMA3) kleuringstoets en die “terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL)" toets, word ook in meer besonderhede aangebied. HOOFSTUK 3 Resultate wat verkry is tydens hierdie studie word hier uiteengesit. Resultate behels die voorkomsyfer van abnormale DNS en die assosiasie met sperm morfologie, spesifiek in die p-patroon en g-patroon. Verdere resultate sluit die korrelasie van sperm morfologie en sperm DNS met bevrugting in vitro, embriokwaliteit en swangerskap uitkomste in. Die persentasie CMA3 positiewe sperme (abnormale DNS) en persentasie TUNEL positiewe sperme (abnormale DNS) het 'n betekenisvolle negatiewe assosiasie met normale sperm morfologie getoon. P-patroon en g-patroon morfologie groepe het betekenisvol van mekaar verskil vir beide CMA3 en TUNEL. 'n Betekenisvolle positiewe assosiasie is tussen CMA3 en TUNEL waargeneem. Geen assosiasie is tussen die persentasie normale sperm morfologie, persentasie CMA3 positiewe sperme en persentasie TUNEL positiewe sperme en IUI swangerskap uitkomste waargeneem nie. 'n Betekenisvolle negatiewe assosiasie is tussen die persentasie TUNEL positiewe sperme en IVB/ICSI swangerskap uitkomste vasgestel. Die persentasie CMA3 positiewe sperme het 'n betekenisvolle positiewe (onverwags) assosiasie met IVB/ICSI swangeskap uitkomste opgewys. Daar was geen assosiasie tussen die drie veranderlikes en IVB/ICSI bevrugting nie. 'n Betekenisvolle positiewe assosiasie is tussen die persentasie normale sperm morfologie en IVB/ICSI embryo kwaliteit waargeneem. Daar was 'n betekenisvolle positiewe assosiasie tussen die persentasie CMA3 positiewe sperme en IVB/ICSI embrio kwaliteit (onverwags). Die persentasie TUNEL positiewe sperme het 'n negatiewe assosiasie met IVB/ICSI embrio kwaliteit getoon. HOOFSTUK 4 Interpretasie van die resultate en toekomstige vooruitsigte is bespreek. Die CMA3 kleuringstoets en TUNEL toets het 'n beperkte vermoë om tussen die p-patroon en g-patroon morfologie groepe te onderskei. P-patroon spermatozoa sal heel waarskynlik oor swakker chromatien verpakking en meer DNS fragmentasie beskik. Sommige p-patroon pasiënte mag egter normale DNS toon, terwyl g-patroon pasiënte abnormale DNS het. Die implementering van 'n sperm DNS toets in andrologie laboratoriums, vir die kliniese diagnose van sperm DNS skade in pasiënte, word aanbeveel.