In vitro produced (IVP) embryos not hatching from the zona pellucida (ZP) after transfer is one possible contributing factor of a lower pregnancy rate when compared to in vivo embryos. This study evaluates using a microscope objective mounted laser to cut the ZP prior to transfer into the recipient to assist hatching. The preliminary data evaluated the effect of laser treatment on IVP embryos and subsequent blastomere survival. In six replicates, bovine oocytes were in vitro produced according to the standard laboratory procedures of TransOva Genetics, Sioux Center, IA. On days 5, 6, and 7 of in vitro culture, embryos were randomly divided into 3 groups: no treatment (Control; n=63), sham ZP cut (Sham; n=68), or ZP cut (Cut; n=70). Control embryos were immediately returned to the incubator. Sham embryos were exposed to all conditions as Cut except laser assisted hatching. The XyClone® system was used to treat the Cut group using pulse strength of 90% and pulse length of 600 μsec. Embryos were returned to culture until day 8 when embryonic development and the percentage of live cells were determined and analyzed with Chi square. The number of developing embryos and the percentage of live cells per embryo showed no significant difference. Mean live cells ranged from 89-96% regardless of day of treatment. The laser assisted hatching effect on IVP embryo viability was evaluated by randomly dividing commercially produced embryos obtained from TransOva Genetics into two groups on day of transfer, Control or Cut. The ZP of treated embryos were cut with the laser using 80% pulse strength and pulse length of 500 μsec on day 7, immediately prior to transfer into estrous synchronized recipients. Ultrasonagraphy determined pregnancy rates. Thirty day pregnancy rates were 49.2% and 54.1% for Control (n= 189) and Cut (n=148) embryos, respectively, and were not statistically different (p > 0.05). However, 60 day Control pregnancy rate was 45.7% (n= 166) and the Cut group rate was 57.7% (n= 123) revealing a statistical difference (p < 0.05). These results demonstrate that the XyClone® system assisted hatching can improve 60 day pregnancy rates for IVP embryos by approximately 11 %.

In vitro produced (IVP) embryos not hatching from the zona pellucida (ZP) after transfer is one possible contributing factor of a lower pregnancy rate when compared to in vivo embryos. This study evaluates using a microscope objective mounted laser to cut the ZP prior to transfer into the recipient to assist hatching. The preliminary data evaluated the effect of laser treatment on IVP embryos and subsequent blastomere survival. In six replicates, bovine oocytes were in vitro produced according to the standard laboratory procedures of TransOva Genetics, Sioux Center, IA. On days 5, 6, and 7 of in vitro culture, embryos were randomly divided into 3 groups: no treatment (Control; n=63), sham ZP cut (Sham; n=68), or ZP cut (Cut; n=70). Control embryos were immediately returned to the incubator. Sham embryos were exposed to all conditions as Cut except laser assisted hatching. The XyClone® system was used to treat the Cut group using pulse strength of 90% and pulse length of 600 μsec. Embryos were returned to culture until day 8 when embryonic development and the percentage of live cells were determined and analyzed with Chi square. The number of developing embryos and the percentage of live cells per embryo showed no significant difference. Mean live cells ranged from 89-96% regardless of day of treatment. The laser assisted hatching effect on IVP embryo viability was evaluated by randomly dividing commercially produced embryos obtained from TransOva Genetics into two groups on day of transfer, Control or Cut. The ZP of treated embryos were cut with the laser using 80% pulse strength and pulse length of 500 μsec on day 7, immediately prior to transfer into estrous synchronized recipients. Ultrasonagraphy determined pregnancy rates. Thirty day pregnancy rates were 49.2% and 54.1% for Control (n= 189) and Cut (n=148) embryos, respectively, and were not statistically different (p > 0.05). However, 60 day Control pregnancy rate was 45.7% (n= 166) and the Cut group rate was 57.7% (n= 123) revealing a statistical difference (p < 0.05). These results demonstrate that the XyClone® system assisted hatching can improve 60 day pregnancy rates for IVP embryos by approximately 11 %.

MSCAGR (Animal Science) / Department of Animal Science / The use of in vitro culture media and assisted hatching techniques remain a challenging obstacle to hatching of blastocyst-stage embryos. Mechanical, chemical, enzymatic thinning and laser assisted techniques have been used previously, but there is still a lack of information on its application and implication in livestock. The aim of this study was to compare the effect of two in vitro culture media ((Ham’s F10 and Tissue Culture Medium 199 (TCM-199)) and four assisted hatching techniques (mechanical, chemical, enzymatic and laser) on blastocyst formation and hatching rate using murine embryos as a model. The C57BL/6 and BALB/c mouse breeds were bred and raised until they reach maturity and then bred naturally to produce a hybrid F1 generation. The light in the breeder house was controlled at 14 hours light and 10 hours darkness. Feed and water were provided ad libitum for the mice. Mature female mice were super-ovulated using equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). A total of 400 blastocysts were collected from the F1 generation and these were allocated equally for the four assisted hatching techniques (laser, mechanical, chemical and enzymatic) as well as a non-treated control group. The blastocysts were paired into a group of 10 and replicated 4-four times for each assisted hatching techniques and control group. The embryos were then cultured for 24 hours and the hatching of the embryos were observed. Hatched embryos were stained for blastomere counting. The general linear model (GLM) of statistical analysis software (SAS) version 9.4 was used to analyze the data. Assisted hatching techniques (laser, mechanical, enzymatic and chemical) yielded 46.86±37.12; 51.07±40.19; 39.05±35.83 and 33.32±37.50% of hatching, respectively under in vitro culture in Ham’s F10. There was a significant difference (p<0.05) observed between assisted hatching techniques using Ham’s F10 as culture medium. In the TCM-199, laser, mechanical, enzymatic and chemical assisted hatching techniques yielded 56.25±43.30; 52.55±35.50; 49.16±37.50 and 33.85±35.50%, respectively, with significant differences (p<0.05). However, the hatching rate of embryos for all techniques was higher when in vitro cultured in TCM-199 compared to those cultured in Ham’s F10, and statistically higher than the control group. In conclusion, laser assisted hatching technique is the best of the techniques to use to assist the hatching of murine embryos and TCM-199 is the best of the two in vitro culture media for the hatching percentage.

MSCAGR (Animal Science) / Department of Animal Science / This study determined the effects of in vitro culture media (Ham’s F10 and TCM199) and assisted hatching techniques (laser or mechanical) on mice embryo survival following cryopreservation. Pure strain C57BL/6 (B6) female (50) and strain BALB /c (C) Male (25) mice were crossed to produce F1 generation of females which were injected for follicular growth and super ovulation at 6 weeks of age and from which embryos were produced 21 h later through in vivo fertilization. Embryos were randomly divided into Petri dishes with different culture media, and the development of embryos was assessed until the morula stage. At the morula stage, selected embryos were assisted to hatch using different techniques, and then cryopreserved in liquid nitrogen using the slow freezing method for a period of 1 week. After 1 week of cryopreservation, the embryos were thawed and cultured in the two different in vitro culture media for 72 hours. Thereafter, the numbers of embryos hatched or survived were recorded after 24 h, 48 h and 72 h. Data was analyzed using ANOVA in Minitab Software Version 16 (2010). Significant difference in embryo quality development was observed between in vitro culture media and stage of embryo development (P<0.05). In the TCM-199 in vitro culture medium, embryo quality development yielded 72, 69 and 69% from day 1 to day 3, while in Ham’s F10 embryo quality development yielded 68, 63 and 60% respectively. Relative to the control (18.1%) assisted hatching improved hatchability significantly (P<0.05) in the order laser (23.6%)>, mechanical (20.8%). There was significant (P<0.01) interaction between assisted hatching techniques and evaluation time, whereby laser assisted hatching was most successful at 48 h (42.0%) while mechanical assisted hatching was most successful at 72 h (36.8%). Cryopreservation reduced the embryo survival compared to fresh embryos. In conclusion laser was the best assisted hatching technique, while TCM-199 was the better medium for in vitro culture of embryo. / NRF

Made available in DSpace on 2016-12-08T16:24:14Z (GMT). No. of bitstreams: 1 PGCA13MA112.pdf: 807586 bytes, checksum: b293744892841538c2ea9e0a5ae8325a (MD5) Previous issue date: 2013-07-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The in vitro production of pig embryos progressed significantly in the last decade. However, the embryo growing rates remain variable and with low embryonic quality. The chromosomal abnormalities as a result of high polyspermy rates and the inefficient culture systems are pointed as the main problems. Thus, the aim of this study was to improve the rates of monospermy, and also the hatching rates of in vitro produced porcine embryos. The first study investigated decreasing sperm concentrations combined with different incubation periods during in vitro fertilization (IVF). A total of 829 oocytes were matured and allocated in two experiments. In the first, oocytes were IVF using different sperm concentrations (62.5, 125, 187.5 or 250 x 103 sperm / mL). In the second experiment the oocytes were IVF using 62.5 x 103 sperm / mL pointed in experiment 1, for 2, 3 or 4 hours incubation time. Then the zygotes were cultured during twelve hours to evaluate the fertilization parameters, or during seven days to evaluate embryonic development. A third experiment was performed to evaluate the sperm acrosome reaction according to the incubation period. The higher sperm concentration reduced embryo production from 33.9% to 13.4%. The reduction of the incubation period from 4 to 3 or 2 hours significantly increased the rates of cleavage (42.1 to 71.6% and 73.3%), embryo development (14 to 34.7 and 38.9%) and hatching (25 to 66.7 and 65.7%. Increased sperm concentration and incubation period significantly altered the parameters related to fertilization, substantially impairing the monospermic embryos production. The number of sperm with reacted acrosome increased significantly as incubation time was increased. In the second study we evaluated the assisted hatching of porcine embryos IVP by zona pellucid incision or treatment with pronase. Oocytes were matured and in vitro fertilized with 62.5 x 103 spermatozoa / mL concentration for three hours. The zygotes were cultured for seven days, and then embryonic development and hatching rates evaluated. It was also determined the cell density and cell apoptosis rates of hatched embryos. Both the manual incision or pronase digestion were effective in weakening the ZP of embryos with high cell density. However, it was observed a reduction in the survival rates, and higher apoptosis rates in hatched embryos treated with Pronase. With basis on the data we concluded that a reduction in sperm concentration and incubation period decreases the incidence of polyspermy and improves the embryo development rates. This is probably due to reduction of capacitated /reacted sperm available for fertilization. Also, the weakening of the zona pellucid by incision improves significantly the number and quality of embryos hatched at the end of embryo culture / A produção in vitro de embriões suínos avançou significativamente na última década. Entretanto, os índices de desenvolvimento embrionário continuam inconstantes e a qualidade dos embriões extremamente baixa. A incidência de anormalidades cromossômicas decorrentes das altas taxas de polispermia e a ineficiência dos sistemas de cultivo são apontados como os principais impasses. Desta forma, este estudo buscou melhorar os índices de monospermia, e as taxas de eclosão dos embriões suínos produzidos in vitro. O primeiro estudo investigou a utilização de diferentes concentrações espermáticas e períodos de incubação dos gametas durante a fecundação in vitro (FIV). Para tanto, um total de 829 oócitos foram maturados e destinados à dois experimentos. No primeiro, os oócitos foram FIV utilizando diferentes concentrações espermáticas (62.5, 125, 187.5 ou 250 x 103 espermatozoides/mL). No segundo, os oócitos foram FIV utilizando 62.5 x 103 espermatozoides/mL durante duas, três ou quatro horas. Após a FIV os zigotos foram cultivados por doze horas para análise dos parâmetros da fertilização ou por sete dias para o estudo do desenvolvimento embrionário. Um terceiro experimento avaliou o número de espermatozóides com acrossoma reagido de acordo com o período de incubação. A máxima concentração de espermatozóides reduziu a produção embrionária (de 33.9% para 13.4%). Já a redução do período de incubação de quatro para três ou duas horas, aumentou significativamente as taxas de clivagem (42.1% para 71.6 e 73.3%), de desenvolvimento embrionário (14% para 34.7 e 38.9%) e de eclosão (25% para 66.7 e 65.7%). Tanto o aumento da concentração espermática, como o aumento período de incubação alteraram significativamente a maioria dos parâmetros relacionados a fecundação, prejudicando substancialmente a produção de embriões monospérmicos. O número de espermatozoides com acrossoma reagido aumentou significativamente com o pronlongamento da fecundação. No segundo estudo, investigou-se a indução da eclosão assistida de embriões suínos PIV através da incisão da zona pelúcida ou pelo tratamento com pronase. Os oócitos foram maturados e fecundados in vitro com uma concentração de 62.5 x 103 espermatozóides/mL por três horas. Os zigotos foram cultivados durante sete dias, então, avaliados quanto ao desenvolvimento embrionário e a incidência de eclosão. Determinamos também a densidade celular e os índices de apoptose celular dos embriões eclodidos. Tanto a incisão da zona como a digestão com pronase foram efetivos na fragilização da ZP dos embriões com alta densidade celular. Todavia, constatou-se uma redução da sobrevivência e aumento do índice de apoptose celular dos embriões eclodidos pelo tratamento com pronase. Com base nos dados obtidos concluímos que a redução da concentração de espermatozóides e do período de incubação diminuem a incidência de polispermia e melhoram o desenvolvimento embrionário. Isto é provavelmente devido a redução da disponibilidade de espermatozóides capacitados/reagidos para a fecundação. Da mesma forma, a fragilização da zona pelúcida através da incisão, melhora significativamente o número e a qualidade dos embriões eclodidos ao final do cultivo embrionário