Effect of various antioxidants on ovarian tissue vitrification success in sheep.

BACKGROUND: Vitrification is a technique of cryopreservation that has been proposed as a promising alternative method for the preservation of oocytes, embryos and gonadal tissue. OBJECTIVE: To determine the effect of different antioxidants on post-thaw viability, morphology of retrieved oocytes and histology of vitrified ovarian tissue. MATERIALS AND METHODS: Four different antioxidants [i.e., resveratrol (20 uM), ZnSO4 (500 uM), curcumin (25 uM) and quercetin (1 uM)] were evaluated after their addition to the vitrification and warming media for their effects on the viability and morphology of retrieved oocytes and the histology of vitrified ovarian tissue. RESULTS: The number of oocytes retrieved from ovarian tissue from the above mentioned antioxidants and vitrified control were 34, 41, 26, 31 and 46 respectively. Among these the number of viable oocytes were found to be 24 (70.6%), 30 (73.1 %), 20 (76.9%), 26 (83.9%) and 33 (71.7%) and the number of oocytes found morphologically normal were 24 (70.6%), 26 (63.4%), 18 (69.2%), 21 (67.7%) and 34 (73.9%) for the above mentioned different antioxidants and vitrified control, respectively. Non-significant (P. > 0.05) differences were found between different treatment groups. Histomorphological evaluation of the ovarian cortical tissue showed that the percentage of intact follicles was significantly (P < 0.05) higher in the fresh control (84.19±3.9) than in other groups. Non-significant differences were found between resveratrol (50.2±5.5), curcumin (48.7±5.7), quercetin (51.6±4.8) and the vitrified control (42.7±6.1) groups; however, the ZnSO4 supplemented group (23.1±8.54) differed significantly (P < 0.05) from other antioxidant groups but was non-significant (P > 0.05) with the vitrified control group (42.7±6.1). CONCLUSION: The addition of antioxidants resveratrol, curcumin and quercetin at these concentrations tended to non-significantly improve the follicular integrity after vitrification. Doi.org/10.54680/fr24410110212.

Strip pulled straw: cost effective alternative cryodevice for the vitrification of oocytes.

BACKGROUND: Increased cooling and warming rates by using a suitable cryodevice allows the use of lower cryoprotectant concentration and reduces cryoinjuries as a result of the rapid passage through the 'dangerous' temperature zone. OBJECTIVE: To evaluate the effectiveness of newly customized strip pulled straw (SPS) with respect to post warming quality, viability, and in vitro maturation for immature oocytes post-vitrification of. MATERIALS AND METHODS: SPS was prepared using conventional French mini straw to combine the merits of OPS and the Cryotop system. Immature sheep oocytes were treated in 15% EG + 15% DMSO, loaded on SPS and plunged into liquid nitrogen (LN). Post thaw quality, viability, and maturation rates of oocytes were determined after 1 week storage in LN. RESULTS: SPS achieved a post-thaw morphological survival of 90.9% with 9.0% morphological defects, 96.4% viability and 51% in vitro maturation. In comparison to OPS, SPS had higher post thaw survival (86.5% vs 67.9%) and maturation rate (57.7% vs 50.5%) with lower morphological defects (13.5% vs 32.1%). Cumulus cell loss was the highest among morphological abnormalities of post warm oocytes in SPS (40.9%) and OPS (44.1%). The data showed acceptable post thaw survival, viability and in vitro maturation rate of immature ovine oocytes using SPS as compared to traditional OPS. CONCLUSION: SPS can be used as a cost effective alternative device for oocyte vitrification. Doi: 10.54680/fr23410110212.

Effect of ovarian storage temperature and time on post thaw viability and maturation rate of vitrified immature oocytes in sheep.

BACKGROUND: Vitrification of oocytes as a method of cryopreservation is quite successful, although it is still being standardized because of structural and molecular sensitivity of oocytes to the cooling and freezing process. OBJECTIVE: To investigate the effect of ovarian storage temperature and time on post thaw viability and maturation rate of vitrified immature oocytes in sheep. MATERIALS AND METHODS: The work consisted of oocyte collection from ovaries of abattoir sheep stored at various temperature (0 degree C, 4 degree C and 25 degree C) and time (0 h, 6 h, 12 h and 24 h) combinations and post thaw viability and in vitro maturation rate evaluation. Vitrification was done in 30% vitrification solution, using ethylene glycol and DMSO, with post vitrification evaluation after 1 week of storage. RESULTS: Significantly higher post thaw viability was observed after storage at 0 degree C for 6 h (95.3%) followed by 12 h (85%), with lowest value at 24 h (66.7%). However at 4 degree C and 25 degree C, values were non-significantly higher after 6 h (96.5 and 100% respectively) followed by 12 h (93 and 100%), with significantly lower values after 24 h (85.7 and 90.7%). At storage temperatures of 25 degree C and 4 degree C, a significantly higher percentage of mature oocytes was observed after 6 h (40 and 39.1%), 12 h (37.3 and 38.1%) and 24 h (34.6 and 36.4%) storage times compared to that at 0 degree C (20.3% at 6 h, 14.2% at 12 h and only 13.8% at 24 h). However, at all storage temperatures, there was a tendency for the level of mature oocytes to decrease with storage time, and the levels were significantly lower than the control. CONCLUSION: Acceptable post thaw viability and in vitro maturation rates for oocytes is maintained up to 24 h in ovaries stored at 4 degree C and 25 degree C compared to at 0 degree C, and these conditions may be used for the storage of ovaries meant for oocyte preservation. doi.org/10.54680/fr22510110412.

Recent advancements in vitrification cryodevices for gamete and gonadal tissue.

Cryopreservation of gametes and gonadal tissue is nowadays primarily accomplished through vitrification. Variables such as cooling rate, viscosity and volume of vitrification solution are critical in gamete vitrification. In addition, sample size and stepwise exposure are also crucial for gonadal tissue vitrification. Recently a class of cryodevices has been developed to reduce the volume of vitrification solution so as to achieve higher cooling rates. Vitrification devices are classified as "open" or "closed" depending on whether the medium comes into direct contact with liquid nitrogen during the process. Examples of the open cryodevices for gamete vitrification are Cryotop, Cryolock, open pulled straw (OPS), etc., and closed devices are Vitrisafe, CryoTip, and high security vitrification kit. Similarly, for tissue vitrification open cryodevices used are needles, cryovials and closed devices used are Cryotissue, ovarian tissue cryosystem, etc. Among all the gamete cryodevices, Cryotop is unique and the best-selling micro-volume storage device. Use of this device has resulted in the highest number of babies born after embryo or oocyte vitrification. Another novel device, Kitasato vitrification system, is a vitrification solution absorber, which is similar to Cryotop but differs in one way, as it possesses a porous membrane that absorbs extra solution from the gamete. This review provides an update on the recent use of cryodevices for gamete and gonadal tissue vitrification. doi.org/10.54680/fr22310110112.

Oocyte and embryo preservation in wild animals: an update.

The reduction in population genetic diversity due to inbreeding depression and the negative impact of human activity on habitats ultimately generates an extinction debt. Therefore, there is always a dire need to save wild population and to protect biodiversity. Preservation of wildlife female germplasm, i.e., oocytes and embryos, is a promising biotechnological tool to conserve species' biodiversity. Other applied tools of Assisted Reproductive Technology (ART) which assure conservation of endangered species include artificial insemination (AI), embryo transfer technology (ETT), and sperm cryopreservation. Only a few studies show the possibility of adapting the cryopreservation techniques developed for domestic animal female genetic material for use with wild animals. Difficulty is encountered in getting samples, accesses to animals for study, and the standardization of protocols for cryopreservation of such genetic material. Our meta-analysis of the literature (published or in press) and on-going studies found that biobanking for the preservation of vital tissues of wild animals is possible. Somatic tissue sections, ovarian tissues, sperms, oocytes and embryos are potential materials for preservation by vitrification. As vitrification is economical and easily applied, it appears to the best option currently available for the preservation of wildlife female genetics in order to conserve species' biodiversity.

Post thaw quality and viability of vitrified immature sheep oocytes using different cryoprotectant concentrations.

BACKGROUND: The cryopreservation of oocytes through vitrification is quite successful but oocyte vitrification is still being standardized because of their structural and molecular sensitivity to the cooling and freezing processes. OBJECTIVE: To evaluate the effect of different cryoprotectant concentrations on post-thaw morphology and viability of immature oocytes in sheep. MATERIALS AND METHODS: Vitrification was achieved in three vitrification solutions comprised of different concentrations of the cryoprotectants ethylene glycol + DMSO, viz., (G1) 20%, (G2) 30%, (G3) 40% ethylene glycol + DMSO in equal ratio. Cryopreservation was in open pulled straws. Post vitrification evaluation was done after 1 week's storage in liquid nitrogen based on morphological evaluation and viability using trypan blue dye. RESULTS: The present study revealed non-significantly higher morphologically normal oocytes in G3 (74.7%) followed by G2 (70.3%), and the lowest in G1 (66.6%). Morphological defects were observed in 33.3 %, 29.6% and 25.2% of oocytes after cryopreservation in 20% (G1), 30% (G2) and 40% (G3) vitrification solutions, respectively. The results were non-significantly different between vitrification solution groups. However, the viability of post thaw immature oocytes was 95.6%%, 84.4% and 81.1% after vitrification in G1 (20%), G2 (30%) and G3 (40%), with viability being significantly highest (P<0.05) in G1 (20%) and lowest in G3 (40%). CONCLUSION: Cryoprotectant concentrations enable the maintenance of normal morphology and minimize cryoinjury during vitrification of immature oocytes.

Effect of different egg yolk-based extenders on the quality of ovine cauda epididymal spermatozoa during storage at 4°C.

Cauda epididymal spermatozoa were obtained from testicles collected from abattoir(s). The pooled sperm samples were divided into four aliquots. Each aliquot was washed separately with the buffer of respective extender and finally extended with the four extenders viz. egg yolk-citrate (EYC), egg yolk-citrate-fructose (EYCF), Tris-citric acid-egg yolk-fructose (TCEYF) and egg yolk-Mcillvaine glucose (EYMG) and preserved at 4°C. The per cent sperm motility for EYC, EYCF, TCEYF and EYMG at 0 h was 50.83%, 56.67%, 75.00% and 31.67%, respectively, and at 72 h was 24.17% (EYC), 30.83% (EYCF), 51.67% (TCEYF) and 7.50% (EYMG). The corresponding figures for live sperm count at 0 h was 83.17%, 86.33%, 90.42% and 81.75% and at 72 h was 64.75%, 73.92%, 76.00% and 57.67%. The corresponding figures for mean per cent intact acrosome at 0 h was 95.33%, 95.50%, 90.92% and 97.25% and at 72 h was 86.17%, 83.92%, 77.58% and 86.33%. The sperm motility was significantly (p < 0.05) higher for TCEYF at different h of preservation from 0 h through 72 h. The sperm motility, live sperm count and per cent intact acrosome declined significantly (p < 0.05) with the advancement of storage time in all the four extenders. Our study concluded that TCEYF was best out of the extenders studied for preservation of cauda epididymal spermatozoa after double centrifugation and extension at 4°C up to 72 h of preservation. However, EYCF also has better potential for the preservation of cauda epididymal spermatozoa as viability was in close proximity and acrosomal integrity was higher compared with TCEYF extender.

Effect of transportation temperature on the quality of cauda epididymal spermatozoa of ram.

The objective of this study was to develop a protocol for ram epididymal sperm preservation that could be applied to wild ruminants for collection and preservation of spermatozoa from dead or hunted animals. Ram testicles collected from abattoirs were used to study the effect of two transportation temperatures viz. ambient temperature (AT) and refrigeration temperature (RT) on the cauda epididymal sperm quality at recovery and during preservation up to 72h at 4°C. For AT the testicles were transported in normal saline in a container (17.9-21.5°C) where as for RT the testicles were transported in an ice-chest (4.9-6°C). The results of the current study revealed that intact acrosome was significantly higher (P<0.01) and other quality parameters like sperm motility, live sperm count, sperm concentration and major sperm abnormalities were also higher (P>0.05) for RT than AT. The mean percent sperm motility for RT and AT was 81.67% and 78.33%, respectively. The corresponding figures were 92.08% and 90.46% for mean live sperm, 98.33% and 90.50% for intact acrosome, 0.50% and 0.33% for major sperm defects. The percent minor abnormality was 79.50% for RT and 77.67% for AT. The most prevalent minor defect was distal cytoplasmic droplet (70-80%). The mean sperm motility for RT and AT at 0h was 82.50% and 75.00%, respectively and the corresponding values at 72h of preservation were 60.00% and 45.83%. The mean live sperm at 0h for RT and AT were 92.92% and 88.92%, respectively and the corresponding figures at 72h were 81.50% and 73.17%. The mean intact acrosome at 0h for RT and AT was 98.58% and 90.58%, respectively and at 72h the corresponding values were 91.66% and 82.25%. The sperm motility, live sperm count and intact acrosome decreased significantly (P<0.05) from 0h to 72h of preservation for both transportation temperatures. The sperm motility, live sperm count and intact acrosome also varied significantly between the transportation temperatures. The major sperm abnormality for both RT and AT at each hour of preservation up to 72h was less than 0.5%. The study concluded that epididymides or testicles should be transported to the laboratory at RT (4.9-6°C) either in an ice-chest or portable refrigerator for their processing, evaluation and storage.