Effects of exposure to dmso in vitrification solution on cytotoxicity and in vitro viability of immature bovine oocyte.

Based on previous studies for vitrification of oocytes, it has been shown that short term exposure to DMSO during vitrification could improve the maturation rate and cause not spontaneous parthenogenesis (Isachenko et al., 2006). In addition, it was reported that DMSO in freezing media caused disassembly of microfilaments and chromosomal abnormalities in mouse oocytes (Vincent et al., 1990). On the other hand, DMSO is categorized as a potent glass former and its existence in vitrification solution seems necessary. The aim of this study was to determine the in vitro viability of immature bovine oocytes vitrified by short or long time exposure to DMSO. Materials and Methods: Cumulus oocytes complexes (COCs) with homogenous ooplasm were recovered from slaughterhouse ovaries and used in this study. The vitrification protocol was adapted from Kuwayama et al (2005) with minor modifications. Briefly, oocytes were washed twice in holding solution (HS, Hepes-buffered TCM medium supplemented with 20% fetal calf serum, FCS) and kept there for about 15 min. Group of four COCs were incubated in the first vitrification solution (VS1; 7.5% DMSO and 7.5% EG in HS) for 12 min. Equilibration in VS1 was performed in three steps of increasing concentration. First (F) and second (S) steps contained 1/3 and 2/3 of VS1 diluted in HS, and the third (T) step contained only pure VS1. Based on removal of DMSO from each step, five treatment groups were designed: (G1) control, (G2) VS1, (G3) F w/o DMSO, (G4) F+S w/o DMSO, and (G5) F+S+T w/o DMSO. For G3, G4 and G5, similar concentration of EG was added to replace DMSO in VS1. All treatment groups were equilibrated into the second vitrification solution (VS2; 15% DMSO, 15% EG and 0.5M sucrose in HS) for a further 60 sec. Two experiments were performed: (a) cytotoxicity after only exposure, and (b) in vitro viability after vitrification processes. In cytotoxicity test, immature oocytes were directly transferred to the warming solution (WS). In vitrification experiment, oocytes were instantly loaded on a Cryotop device and submerged into liquid nitrogen (LN2) for storage. The time of exposure from VS2 to LN2 was not longer than 90 s. Vitrified samples were maintained in LN2 for at least 10 days. Immediately after removing the Cryotop from LN2, thin strip of Cryotop was submerged in 3 ml HS plus 1M sucrose (WS; 39°C) and smoothly tried to detach oocytes from Cryotop device. Immature oocytes were left in WS for one minute and then transferred to HS plus 0.5M and 0.15M sucrose solution for 3 and 5 min, respectively. Finally, the immature oocytes were washed twice in HS for 5 min each and processed for in vitro maturation. Significant differences among treatments used in the experiment were revealed by one-way analysis of variance and followed by Duncan's multiple range test for mean comparisons (P < 0.05) using SAS software (ver. 9.1)

Cryotop and development of vitrified immature bovine oocytes

The effectiveness of different cryodevices (open-pulled straw (OPS), electron microscopy grid (EMG), and Cryotop was evaluated for vitrification of immature bovine oocytes. Polar body, metaphase II stage (MII), survivability, and subsequent developmental rates were determined. Only oocytes with four or five layers of cumulus cells were used. Oocytes were equilibrated in two vitrification solutions - 1: 10% DMSO + 10% ethylene glycol (EG) for 30-45sec and 2: 20% DMSO + 20% EG +0.5M sucrose for 25sec -, mounted on one of the cryodevices and directly plunged into liquid nitrogen for 10 days. Immature vitrified oocytes using Cryotop showed the highest rates of polar body extrusion (PB) and nuclear maturity (MII); 41 and 58% respectively. Vitrified oocytes using OPS and EMG showed 26 and 32%; and 35 and 46% of PB and MII rates, respectively. The highest survivability resulted from Cryotop and EMG groups and no significant difference was found between them. Vitrified oocytes using Cryotop had the highest cleavage and blastocyst rates. All of the mean rates for vitrified immature oocytes were significantly lower than that of control group (P<0.05). The results of this study showed the superiority of Cryotop device for vitrification of immature bovine oocyte

Structural Changes in Cattle Immature Oocytes Subjected to Slow Freezing and Vitrification

This study was conducted to evaluate the effect of different cryopreservation methods (slow-freezing and vitrification) on structural changes of bovine immature oocytes. Bovine ovaries were collected from local abattoirs. Cumulus-oocyte-complexes (COCs) were retrieved using aspiration method from 2-6 mm follicles. In Experiment 1, selected oocytes were randomly divided into 4 treatment groups namely freezing solution-exposed, frozen-thawed, vitrification solution-exposed and vitrified-thawed and then oocytes abnormalities were examined under a stereomicroscope. In Experiment 2, oocytes were randomly allocated to the same grouping as experiment 1 plus control group. Following freezing or vitrification, all oocytes were fixed in glutaraldehyde and processed for transmission electron microscopy. In experiment 1, there was a higher incidence of abnormalities in the frozen-thawed and vitrified-warmed oocytes compared to those in freezing solution and vitrification solution-exposed groups (P<0.05). In experiment 2, there were marked alterations in the perivitelline space, microvilli and vesicles of frozen-thawed and vitrified-warmed oocytes characterized by loss of elasticity and integrity of cytoplasmic processes and microvilli following cooling and warming. In conclusion, ethylene glycol-based freezing and vitrification solutions are suitable choices for cryopreservation of immature oocytes and most organelles are able to retain their normal morphology following cryopreservation and thawing processes

Collection, analysis and cryopreservation of semen from Malayan gaur (Bos gaurus hubbacki): A preliminary study

The Malayan gaur (&lt;i&gt;Bos gaurus hubbacki&lt;/i&gt;) or Seladang is classified as vulnerable by the International Union for Conservation of Nature and Natural Resources (IUCN). The Malayan gaur is mainly distributed in the tropical woodlands of Peninsular Malaysia and Southern Thailand. The aim of this study was to collect, analyze and cryopreserve the semen of wild Malayan gaur. Transrectal massage (TM) and electroejaculation (EEJ) technique was applied in semen collection of the Malayan gaur. The semen was then cryopreserved in liquid nitrogen using slow freezing technique. Makler counting chamber was used to evaluate sperm concentration and motility, while the sperm viability and morphology of fresh and post-thaw sperm was determined using eosin-nigrosin staining protocol. As a result, we have successfully collected the Malayan gaur semen using EEJ technique. Sperm motility, viability and morphological changes of the post-thaw semen of Malayan gaur were found undesirable due to the complication of the cryopreservation process. On the basis of current study it can be concluded that Malayan gaur bulls semen can be obtain by EEJ with no evidence of rectal trauma.  Optimization of the process of cryopreservation for Malayan gaur sperm is needed to maintain the cryoviability of the good sperm quality. The data generated in this study would be useful in conservation of genetic diversity program for Malayan gaur.Key words: Cryopreservation, Malayan gaur, Semen collection

Collection, analysis and cryopreservation of semen from Malayan gaur (Bos gaurus hubbacki): A preliminary study

The Malayan gaur (Bos gaurus hubbacki) or Seladang is classified as vulnerable by the International Union for Conservation of Nature and Natural Resources (IUCN). The Malayan gaur is mainly distributed in the tropical woodlands of Peninsular Malaysia and Southern Thailand. The aim of this study was to collect, analyze and cryopreserve the semen of wild Malayan gaur. Transrectal massage (TM) and electroejaculation (EEJ) technique was applied in semen collection of the Malayan gaur. The semen was then cryopreserved in liquid nitrogen using slow freezing technique. Makler counting chamber was used to evaluate sperm concentration and motility, while the sperm viability and morphology of fresh and post-thaw sperm was determined using eosin-nigrosin staining protocol. As a result, we have successfully collected the Malayan gaur semen using EEJ technique. Sperm motility, viability and morphological changes of the post-thaw semen of Malayan gaur were found undesirable due to the complication of the cryopreservation process. On the basis of current study it can be concluded that Malayan gaur bulls semen can be obtain by EEJ with no evidence of rectal trauma. Optimization of the process of cryopreservation for Malayan gaur sperm is needed to maintain the cryoviability of the good sperm quality. The data generated in this study would be useful in conservation of genetic diversity program for Malayan gaur