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

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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.

Preservation of bull semen at sub-zero temperatures /

Cover title.

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.

Efeito da adição de colesterol sobre a viabilidade e fertilidade de espermatozóides equinos refrigerados

Pós-graduação em Medicina Veterinária - FMVZ

Efeito da incorporação do glicerol sobre a viabilidade e fertilidade do sêmen bovino refrigerado

Pós-graduação em Medicina Veterinária - FMVZ

Qualidade espermática após seleção por centrifugação em Ovipure® ou gradiente de Percoll® do sêmen ovino criopreservado

Pós-graduação em Biotecnologia Animal - FMVZ

Indução a espermiação e criopreservação espermática de Brycon cephalus (Gunther, 1869) (teleostei: characidae)

Pós-graduação em Ciência e Tecnologia Animal - FEIS

Características seminais e resfriamento de sêmen de tamanduá (Myrmecophaga tridactyla) de vida livre

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Características físicas e morfológicas do sêmen de Bos taurus e Bos indicus antes e após a criopreservação

Pós-graduação em Medicina Veterinária - FCAV

Ultrastructure, osmotic tolerance, glycerol toxicity and cryopreservation of caput and cauda epididymidal kangaroo spermatozoa

The aim of the present study was to compare cryopreservation, osmotic tolerance and glycerol toxicity between mature and immature epididymal kangaroo spermatozoa to investigate whether the lack of cryopreservation success of cauda epididymidal spermatozoa may be related to the increased complexity of the sperm ultrastructure acquired during epididymal transit. Caput and cauda epididymidal spermatozoa were recovered from red-necked wallabies (RNW; Macropus rufogriseus) and eastern grey kangaroos (EGK; M. giganteus). In Experiment 1, caput and cauda epididymidal spermatozoa were frozen and thawed using a standard cryopreservation procedure in Triscitrate buffer with or without 20% glycerol. Although cryopreservation of caput epididymidal spermatozoa resulted in a significant increase in sperm plasma membrane damage, they were more tolerant of the procedure than spermatozoa recovered from the cauda epididymidis (P< 0.05). In Experiment 2, caput and cauda epididymidal EGK spermatozoa were diluted into phosphate-buffered saline media of varying osmolarity and their osmotic tolerance determined. Plasma membranes of caput epididymidal spermatozoa were more tolerant of hypo-osmotic media than were cauda epididymidal spermatozoa ( P< 0.05). In Experiment 3, caput and cauda epididymidal RNW spermatozoa were incubated in Tris-citrate buffer with and without 20% glycerol at 35 and 4 degrees C to examine the cytotoxic effects of glycerol. At both temperatures, caput epididymidal spermatozoa showed less plasma membrane damage compared with cauda epididymidal spermatozoa when exposed to 20% glycerol ( P< 0.05). These experiments clearly indicate that epididymal maturation of kangaroo spermatozoa results in a decreased ability to withstand the physiological stresses associated with cryopreservation.

Freezing process optimisation of pharmaceutical formulations for freeze drying

The body of work presented in this thesis are in three main parts: [1] the effect of ultrasound on freezing events of ionic systems, [2] the importance of formulation osmolality in freeze drying, and [3] a novel system for increasing primary freeze drying rate. Chapter 4 briefly presents the work on method optimisation, which is still very much in its infancy. Aspects of freezing such as nucleation and ice crystal growth are strongly related with ice crystal morphology; however, the ice nucleation process typically occurs in a random, non-deterministic and spontaneous manner. In view of this, ultrasound, an emerging application in pharmaceutical sciences, has been applied to aid in the acceleration of nucleation and shorten the freezing process. The research presented in this thesis aimed to study the effect of sonication on nucleation events in ionic solutions, and more importantly how sonication impacts on the freezing process. This work confirmed that nucleation does occur in a random manner. It also showed that ultrasonication aids acceleration of the ice nucleation process and increases the freezing rate of a solution. Cryopreservation of animal sperm is an important aspect of breeding in animal science especially for endangered species. In order for sperm cryopreservation to be successful, cryoprotectants as well as semen extenders are used. One of the factors allowing semen preservation media to be optimum is the osmolality of the semen extenders used. Although preservation of animal sperm has no relation with freeze drying of pharmaceuticals, it was used in this thesis to make a case for considering the osmolality of a formulation (prepared for freeze drying) as a factor for conferring protein protection against the stresses of freeze drying. The osmolalities of some common solutes (mostly sugars) used in freeze drying were determined (molal concentration from 0.1m to 1.2m). Preliminary investigation on the osmolality and osmotic coefficients of common solutes were carried out. It was observed that the osmotic coefficient trend for the sugars analysed could be grouped based on the types of sugar they are. The trends observed show the need for further studies to be carried out with osmolality and to determine how it may be of importance to protein or API protection during freeze drying processes. Primary drying is usually the longest part of the freeze drying process, and primary drying times lasting days or even weeks are not uncommon; however, longer primary drying times lead to longer freeze drying cycles, and consequently increased production costs. Much work has been done previously by others using different processes (such as annealing) in order to improve primary drying times; however, these do not come without drawbacks. A novel system involving the formation of a frozen vial system which results in the creation of a void between the formulation and the inside wall of a vial has been devised to increase the primary freeze drying rate of formulations without product damage. Although the work is not nearly complete, it has been shown that it is possible to improve and increase the primary drying rate of formulations without making any modifications to existing formulations, changing storage vials, or increasing the surface area of freeze dryer shelves.