Factors affecting fertility in frozen-thawed boar sperm

Frozen-thawed boar sperm holds the potential to have an impact on the future of the swine industry. Utilization of this technology could improve a swine producer’s ability to access top-tier genetics from around the world, to improve efficiency, profitability, and the quality of product to meet consumer demands. Effective application of frozen-thawed sperm can help reduce the potential risk associated with devastating economic loss due to the spread of disease. Frozen storage of boar sperm also provides a safeguard in the event of disease outbreaks, as genetic material from paternal lines can be preserved and banked for repopulation purposes. Historically these benefits have been masked by reduction in fertility measures such as litter size. The reduced fertility results from the damage sustained by the sperm cell during cryopreservation. However, increased understanding of this damage has lead to improved cryopreservation methods, ultimately increasing post-thaw viability and fertility. Enhancements in breeding technology have also resulted in a better understanding of the AI methods required to achieve acceptable farrowing rates and litter size. Fertility following AI with frozen-thawed sperm is approaching that of liquid stored sperm, and producers may soon reap the benefits of this technology. This thesis will outline the current swine industry, opportunities for utilizing frozen-thawed sperm, the main components of sperm, why they are susceptible to damage, and current freezing and breeding practices. Objective 1 was to develop a cryopreservation protocol for our lab that resulted in consistent post-thaw motility ( ≥ 40%) that would eventually be used by Illinois boar studs for domestic and international sale of frozen sperm. Evaluation with both manual microscopy and CASA methods were conducted to verify quality. A preliminary breeding trial was then conducted to test the fertility of sperm frozen with this method. There were 41 ejaculates from 23 boars used for freezing. Sperm were frozen at 1.4x109 sperm/mL, averaging 55.61.1% (meanSE) motility, following thaw. The samples assessed were not different (P>0.05) in motility when compared with manual or CASA systems, and results were most reliable at a 1:40 sperm dilution. In the preliminary breeding trial, gilts (n=14) were inseminated with either a single (n=10) or double (n=4) AI using 1, 2, or 4x109 motile, frozen-thawed sperm. Overall, the resulting pregnancy rates averaged 71.4% and numbers of normal fetuses per litter averaged 15.51.3 per litter. A feasibility study for freezing cost per ejaculate was estimated at $275/ejaculate or $11/dose of frozen-thawed semen at standard doses of 5x109 total frozen-thawed sperm. This cost estimate did not include genetic value, fixed equipment costs, depreciation, or variable lab space fees. Objective 2 focused on the proper methods for breeding with frozen-thawed boar sperm to achieve fertility. Our hypothesis was that increased numbers of inseminations and increased numbers of motile frozen-thawed sperm would improve pregnancy rate and litter size. Results showed acceptable fertility at high sperm numbers, but also the optimal method for insemination with the lowest dose tested. Gilts (n=111) responded to synchronization methods and were bred with 1, 2, or 4x109 motile frozen-thawed sperm from six boars using a single AI at 32 h, or a double AI, with the first AI at 24 and 32 h following estrus. Ultrasound was conducted at 12 h intervals to estimate the time of ovulation. On day 32 of gestation, overall pregnancy rate (73%) and number of normal fetuses per litter (10.80.5) across all treatments did not differ, and were not affected by number of motile sperm, or the interaction of number of motile sperm and number of inseminations. However, the number of inseminations tended to affect (P=0.14) the number of normal fetuses. Litter size increased with a double AI compared to single AI. Multiple inseminations helped to allow insemination to occur close to ovulation in response to variation in the time of ovulation. Both pregnancy rate and number of normal fetuses were greater when the time of the AI at 32 h occurred closer to the estimated time of ovulation (P<0.05). In addition, other factors such as presence of an abnormal ovary at day 30 decreased (P<0.001) pregnancy rate, while boar affected number of normal fetuses (P<0.01). Analysis of our data using a fertility index revealed doses of 2x109 motile sperm with multiple AI can achieve acceptable fertility with use of less sperm, when compared to AI using 4x109 motile sperm. The methods described here will investigate the potential for improved fertility when using frozen-thawed sperm, while accounting for variation in time of ovulation.

Adição de Ácido Linoleico conjugado (CLA) no diluidor de congelação de sémen de touro, recolhido Post-Mortem, das raças Holstein Frísia, Aberdeen Angus, Charolês e Limousine

Dissertação de Mestrado, Engenharia Zootécnica, 7 de Abril de 2016, Universidade dos Açores.

Sincronização do estro e coleta de embriões em suínos nacionais

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, 2016.

Dose inseminante para fertilização artificial de ovócitos de dourado

Objetivou-se determinar a dose inseminante adequada para uso na fertilização artificial de ovócitos de dourado (Salminus brasiliensis). Os ovócitos foram distribuídos em delineamento inteiramente casualizado, e fertilizados com uma das relações espermatozoides/ovócito 6,0×10³; 6,0×10(4); 6,0×10(5); 6,0×10(6) ou 3,0×10(7), cada uma com quatro repetições. Considerou-se unidade experimental uma incubadora de volume útil de 2,5 L, contendo 2,0 mL de ovócitos não-hidratados. As taxas de fertilização foram mensuradas 8 horas após o início da fertilização. Com intuito de verificar possíveis efeitos da diluição seminal na movimentação dos espermatozoides, realizou-se a mensuração do tempo de duração da motilidade espermática dos espermatozoides de dourado, ativados por meio de diferentes relações de diluição: 6,8×10-5; 6,8×10-4; 6,8×10-3; 6,8×10-2; 3,4×10-1 e 1,0 mL de sêmen por mL de água. O tempo de duração da motilidade foi avaliado em delineamento inteiramente casualizado composto de seis tratamentos e três repetições. As taxas de fertilização apresentaram relação quadrática com o número de espermatozoides por ovócito. As relações de diluição do sêmen tiveram efeito inversamente proporcional sobre a duração da motilidade espermática. A relação que proporcionou melhores taxas de fertilização artificial de ovócitos de dourado (Salminus brasiliensis) foi de 30.722 espermatozoides por ovócio.

Ajuste del tiempo de inseminación con semen sexado en vacas superovuladas

En la actualidad los resultados de la combinación del semen sexado y superovulación (SOV) no han sido muy alentadores debido a la baja cantidad de espermatozoides así como a la corta vida media de los mismos. En un trabajo previo realizado en Brasil en vacas Holstein, se demostró que usando el protocolo de SOV P36/Lh60 e inseminando con semen sexado a las 18 y 30 horas de aplicado el inductor de la ovulación (intervalo de 12 hs entre inseminaciones) se obtiene igual cantidad de estructuras transferibles que inseminando a las 12 y 24 horas con semen no sexado. El objetivo de esta investigación fue ajustar las horas de la inseminación artificial (IA) en vacas superovuladas e inseminadas con semen sexado para lograr mejor sincronía con las ovulaciones y así aumentar la cantidad de embriones transferibles. Este ajuste evaluó 2 momentos de IA en los cuales el intervalo entre ambas se disminuyó a 6 hs. Para esto se usaron 30 vacas Holstein en producción superovuladas con el protocolo P36/LH60 y se dividieron en tres grupos en forma aleatoria; en el grupo IA18/30 (control; n=10) las inseminaciones se realizaron a las 18 horas y 30 hs de la aplicación del inductor de la ovulación (GnRh); en el grupo IA18/24 (n=10) sé inseminó a las 18 y 24 hs de la GnRH y al grupo IA24/30 (n=10) se inseminó a las 24 y 30 (se usó 2.1x106 espermatozoides sexados / inseminación). No se encontró diferencia estadística en los tres grupos, sin embargo el grupo IA18/24 mostró ventaja numérica sobre el grupo IA24/30 y el Control (4,1±1,5 vs 1,3±0,4 y 1,9±0,6 respectivamente) en la cantidad de embriones transferibles, con lo que concluimos que el ajuste en las horas de IA con semen sexado en vacas Holstein superovuladas puede ser usado con resultados similares a los trabajos anteriores

Artificial fertilization of oocytes and sperm activation in pacu: effects of the spermatozoa:oocyte ratio, water volume, and in natura semen preservation

Objetivou-se foi avaliar a fertilização artificial e a duração da motilidade espermática em pacus com diferentes doses inseminantes, volumes de água e preservação do sêmen in natura. Foram realizados quatro experimentos para avaliação do efeito de doses inseminantes (7x10³, 7x10(4), 7x10(5), 7x10(6) e 7x10(7) espermatozoides ovócito-1) sobre a fertilização artificial dos ovócitos; do efeito do volume de água (0,5; 15,0; 30,0; 45,0 e 60,0 mL de água mL-1 de ovócitos) com doses inseminantes de 105.481 e 210.963 espermatozoides ovócito-1; do efeito de diluição do sêmen (0,005; 0,05; 0,5 e 5,0 µL de sêmen mL-1 de água) sobre a duração da motilidade espermática; e do efeito do armazenamento a 15 ºC por 9 h sobre a duração da motilidade espermática e o índice de sobrevivência espermática. Os maiores resultados obtidos foram: doses inseminantes entre 7x10³ e 7x10(7) espermatozoides ovócito-1; 15 a 60 mL de água mL-1 de ovócitos; diluição de 0.005 µL sêmen mL-1 de água e 98,65% de sobrevivência espermática até o tempo de preservação de 2h45min36s. A preservação a 15ºC por 9 horas não influencia a duração da motilidade espermática. As maiores taxas de fertilização podem ser observadas no emprego de 0,27 a 270 µL de sêmen mL-1 de ovócitos, com 15 a 60 mL de água para ativação.

Effects of the spermatozoa: oocyte ratio, water volume and water temperature on artificial fertilization and sperm activation of cascudo-preto

The objective of this study was to evaluate the effects of water volume and water temperature on the sperm motility duration and the number of spermatozoa, and the water volume on the fertilization rates of oocytes of Rhinelepis aspera. Experiments were carried out to evaluate the effect of semen dilutions (1.74×10-5, 1.74×10-4, 1.74×10-3, 1.74×10-2, 1.74×10-1 and 1.00 mL of sperm.mL-1 of water) and water temperature (5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 ºC) on spermatozoa motility duration. In addition, the effects of insemination dose (7×10³, 7×10(4), 7×10(5), 7×10(6) and 7×10(7) spermatozoa.oocyte-1) and water volume (1.0, 30.0, 60.0, 90.0 and 120.0 mL water.2.0 mL-1 oocytes) on the artificial fertilization rates of oocytes were evaluated. The longest sperm motility duration were observed for the semen dilution of 1.74×10-5 mL semen.mL-1 water and in water at 5 ºC. The highest fertilization rates were obtained for insemination doses between 7.00×10³ and 1.23×10(7) spermatozoa. oocyte-1 and water volume of 28.11 mL water.2.0 mL-1 oocytes.

Evaluation of breeding strategies for polledness in dairy cattle using a newly developed simulation framework for quantitative and Mendelian traits

Background: Intensified selection of polled individuals has recently gained importance in predominantly horned dairy cattle breeds as an alternative to routine dehorning. The status quo of the current polled breeding pool of genetically-closely related artificial insemination sires with lower breeding values for performance traits raises questions regarding the effects of intensified selection based on this founder pool. Methods: We developed a stochastic simulation framework that combines the stochastic simulation software QMSim and a self-designed R program named QUALsim that acts as an external extension. Two traits were simulated in a dairy cattle population for 25 generations: one quantitative (QMSim) and one qualitative trait with Mendelian inheritance (i.e. polledness, QUALsim). The assignment scheme for qualitative trait genotypes initiated realistic initial breeding situations regarding allele frequencies, true breeding values for the quantitative trait and genetic relatedness. Intensified selection for polled cattle was achieved using an approach that weights estimated breeding values in the animal best linear unbiased prediction model for the quantitative trait depending on genotypes or phenotypes for the polled trait with a user-defined weighting factor. Results: Selection response for the polled trait was highest in the selection scheme based on genotypes. Selection based on phenotypes led to significantly lower allele frequencies for polled. The male selection path played a significantly greater role for a fast dissemination of polled alleles compared to female selection strategies. Fixation of the polled allele implies selection based on polled genotypes among males. In comparison to a base breeding scenario that does not take polledness into account, intensive selection for polled substantially reduced genetic gain for this quantitative trait after 25 generations. Reducing selection intensity for polled males while maintaining strong selection intensity among females, simultaneously decreased losses in genetic gain and achieved a final allele frequency of 0.93 for polled. Conclusions: A fast transition to a completely polled population through intensified selection for polled was in contradiction to the preservation of high genetic gain for the quantitative trait. Selection on male polled genotypes with moderate weighting, and selection on female polled phenotypes with high weighting, could be a suitable compromise regarding all important breeding aspects.

Preservation of Honey Bee (Apis mellifera L.) Semen

L’abeille domestique (Apis mellifera Linnaeus) joue un rôle crucial comme pollinisateur dans l’industrie de l’agriculture. Cependant, durant les dernières décennies, une mortalité des colonies d’abeilles a été observée partout à travers le monde. La conservation du sperme d’abeille est un outil efficace pour sauvegarder la diversité génétique. Sa conservation est possible à température pièce, mais la cryoconservation serait une meilleure méthode pour la conservation à long terme. Notre objectif général est de développer une méthode de cryoconservation de la semence d’abeille. L’hypothèse no.1 était que la cryoconservation de la semence d’abeille est plus efficace à long terme que les températures au-dessus de 0 °C. Nous avons évalué l’efficacité, basé sur la viabilité des spermatozoïdes, de deux températures de conservation: -196 °C et 16 °C. Après un an de conservation, la semence congelée avait une meilleure viabilité comparée à 16°C (76% ± 5% vs 0%; p < 0,05). Par la suite, la spermathèque des reines inséminées avec la semence cryoconservée a été évaluée par la migration des spermatozoïdes ainsi que la viabilité des spermatozoïdes. Il y avait beaucoup de variabilités dans nos résultats. Nous n’avons pas été en mesure de vérifier si l’ajout de la centrifugation après la conservation améliore la fertilité des reines après insémination. Toutefois, nos résultats confirment que la cryoconservation est une technique efficace pour conserver la semence d’abeille à long terme.

Clínica de animais exóticos

Este relatório de estágio descreve as atividades desenvolvidas no estágio curricular e desenvolve o tema ―Inseminação artificial em Python regius‖. O estágio de seis meses foi realizado na Exoclinic – clínica veterinária de aves e exóticos, permitindo consolidar conhecimentos através da prática clínica e agregar-lhes a área da medicina de animais exóticos, dedicando especial atenção à medicina de répteis. Desenvolvem-se os casos mais frequentes: medicina preventiva nas várias classes de animais, patologia dentária em roedores e lagomorfos, doença do bico e das penas dos psitacídeos e anorexia em répteis. Na monografia aborda-se a anatomofisiologia e o comportamento reprodutivos das cobras e a sua influência no maneio reprodutivo em cativeiro, métodos de avaliação de reprodutores e técnicas de inseminação artificial. Realizou-se um ensaio prático com seis animais, realizando colheitas de sémen, avaliação de alguns parâmetros do sémen, controlo folicular por ecografia e inseminação artificial. Apresentam-se os métodos, os resultados e respetiva discussão; Abstract: Exotic animal medicine This traineeship report describes the activities during the traineeship and develops the theme "Artificial insemination in Python regius." The six-month internship was held in Exoclinic - clínica veterinária de aves e exóticos, consolidating knowledge through clinical practice and adding the medical field of exotic animals, with special attention to reptiles. The most frequent clinical cases are developed: preventive medicine in the various classes of animals, dental pathology in rodents and lagomorphs, Psittacine Beak and Feather Disease in birds and anorexia in reptile. The monograph addresses the anatomy, physiology and reproductive behavior of snakes and their influence on reproductive management in captivity, methods for reproductors selection and evaluation and artificial insemination techniques. Implementation of a field trial with six animals, performing semen crops and evaluation of some of its parameters, follicular monitoring by ultrasound and artificial insemination. Methods and results are presented and discussed.

Cirurgia e clínica de espécies pecuárias

Neste relatório são, primeiramente, descritas as atividades desenvolvidas durante o Estágio Curricular do Mestrado Integrado em Medicina Veterinária da Universidade de Évora, no âmbito de clínica e cirurgia de espécies pecuárias. Pretendeu-se, depois, determinar se a suplementação de bovinos de aptidão leiteira com β-caroteno injetável contribui para reduzir o número de inseminações artificiais necessárias para uma gestação. Por outro lado avaliou-se a influência que podem ter algumas variáveis, como sejam a estação do ano ou o número de partos da vaca. Procurou-se, ainda, estudar a evolução dos teores séricos de β-caroteno nos animais suplementados e não suplementados com Dalmafertyl®, utilizando a técnica de cromatografia líquida de alta eficácia ultravioleta. A suplementação parenteral de β-caroteno, nomeadamente com Dalmafertyl®, na Primavera, mostra resultados benéficos na reprodução de vacas e novilhas de aptidão leiteira, aumentando a produtividade e diminuindo custos; ABSTRACT: LARGE ANIMAL SURGERY AND CLINICS On the following report are described the activities that took place during the Curricular Externship as part of the Veterinary Medicine Integrated Masters from University of Évora, in large animal surgery and clinics. Also, it was intended to determine if injectable β-carotene supplementation on dairy cattle can be a decreasing factor on the artificial insemination number needed to result in pregnancy. On the other hand, it was assessed its influence on some variables that can affect pregnancy, such as seasons and calving number. It has been studied the evolution of β-carotene serum levels on Dalmafertyl® supplemented and non-supplemented animals using the high-performance liquid chromatography ultraviolet detection method, as well. β-carotene parenteral supplementation using Dalmafertyl® seems to have benefic results on dairy cattle reproduction when it comes to Spring, which leads to productivity increase and cost decrease

Cryopreservation of Sheep Produced Embryos – Current and Future Perspectives

Due to economical and scientific limitations, sheep embryo reproductive technologies are less commercially applied than in other animal species. However, it is very clear that, in the near future, those techniques are expected to have a central role in animal production as a consequence of genetic and reproductive demands. One drawback is that results obtained after sheep embryo cryopreservation are unattractive for commercial purposes. It is expected that a successful cryopreservation of sheep embryos can push forward all other reproductive biotechnologies in this species, such as multiple ovulation and embryo transfer (MOET), artificial insemination, or in vitro production of embryos. This paper tries to discuss the current and future perspectives of cryopreservation of in vivo- and in vitro-produced sheep embryos concerning advantages and limitations for its practical use and possible solutions for improving methods to allow a higher survival rate of cryopreserved embryos.

Route of Oxytocin administration and nonsurgical embryo recovery in Santa Inês ewes after induction synchronous estrus.

Oxytocin has been used to promote cervical dilation with the objective to access uterus both in artificial insemination and transcervical embryo recovery in sheep and goats. The objective of this study was to test the effect of two routes of oxytocin administration on nonsurgical embryo recovery efficiency in Santa Inês ewes after induction of synchronous estrus. Results of this study showed that nonsurgical transcervical embryo recovery can be efficiently done in some ewes; a higher number of individuals is needed to conclude that transcervical embryo recovery can be efficiently done in ewes and surgery embryo collections can be avoided in near to 60% of pluriparous Santa Inês ewes; and that the route of oxytocin administration did not affect the parameters evaluated.

Update and overview on assisted reproductive technologies (ARTs) in Brazil.

2016