Lis S. Marques

Viability of zebrafish (Danio rerio) ovarian follicles after vitrification in a metal container.

Cryopreservation of ovarian tissue has been studied for female germline preservation of farm animals and endangered mammalian species. However, there are relatively few reports on cryopreservation of fish ovarian tissue and especially using vitrification approach. Previous studies of our group has shown that the use of a metal container for the cryopreservation of bovine ovarian fragments results in good primordial and primary follicle morphological integrity after vitrification. The aim of this study was to assess the viability and in vitro development of zebrafish follicles after vitrification of fragmented or whole ovaries using the same metal container. In Experiment 1, we tested the follicular viability of five developmental stages following vitrification in four vitrification solutions using fluorescein diacetate and propidium iodide fluorescent probes. These results showed that the highest viability rates were obtained with immature follicles (Stage I) and VS1 (1.5 M methanol + 4.5 M propylene glycol). In Experiment 2, we used VS1 to vitrify different types of ovarian tissue (fragments or whole ovaries) in two different carriers (plastic cryotube or metal container). In this experiment, Stage I follicle survival was assessed following vitrification by vital staining after 24 h in vitro culture. Follicular morphology was analyzed by light microscopy after vitrification. Data showed that the immature follicles morphology was well preserved after cryopreservation. Follicular survival rate was higher (P < 0.05) in vitrified fragments, when compared to whole ovaries. There were no significant differences in follicular survival and growth when the two vitrification devices were compared.

Evaluation of prenucleic acid extraction for increasing sensitivity of detection of virus in bovine follicular fluid pools

Bovine viral diarrhea virus (BVDV), bovine herpesvirus type 1 (BoHV-1), and bovine herpesvirus type 5 (BoHV-5) are major cattle pathogens that can be present in biological materials used in assisted reproduction biotechnologies. The aim of the present study was to increase the sensitivity of the polymerase chain reaction (PCR) for detection of BVDV, BoHV-1, and BoHV-5 in bovine follicular fluid (FF) collected during oocyte retrieval for in vitro embryo production. Ovaries were collected immediately after slaughter at a commercial abattoir, aspirated, and the 7336 samples of FF were pooled in 84 samples. Before testing the FF field samples, sensitivity of the protocol was determined using a prenucleic acid extraction procedure that was directly compared with standard RNA or DNA extraction protocols. The prenucleic acid extraction procedure increased sensitivity of reverse transcription (RT)-PCR for BVDV and nested PCR for BoHV-1 and BoHV-5 by 100 and 10 times, respectively. The 84 FF pools were assayed for BVDV, BoHV-1, and BoHV-5 using virus isolation and RT-PCR or nested PCR. Fourteen (16.7%) FF pools were positive for BVDV RNA, and one (1.2%) was positive for BoHV-1 DNA. Two of the BVDV RT-PCR positive samples and the one BoHV-1 PCR positive sample were also positive in cell culture, demonstrating that FF contained infectious viruses. In this study, the prenucleic acid extraction procedure increased the sensitivity of RT-PCR and PCR detection. This study highlighted the importance of assuring biosecurity by detecting the presence of viral pathogens in biological materials used during in vitro embryo production.

Welfare and stress in fish

Moral philosophy is involved in developing a systematic approach to determine the sense of right and wrong and has been the major subject to define ethic in animal production. Traditionally, from a humancentered perspective, fish are simply objects for human consumption. Welfare is a complex conception of human consciousness about animal suffering. Stress is a consequence of low welfare and causes important changes in animal organisms but also in behavior and life quality. The main aspects of fish welfare approached on this study are: social subordination and stocking density; water quality (temperature and ammonia); transport of live fish; external indicators of welfare; pain perception and fear.