Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep

Summary

The protocol aims at providing a standard method for the vitrification of adult and juvenile sheep oocytes. It includes all the steps from the preparation of the in vitro maturation media to the post-warming culture. Oocytes are vitrified at the MII stage using Cryotop to ensure the minimum essential volume.

Abstract

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily obtained from a slaughterhouse. Adult ovaries always bear several antral follicles, while in pre-pubertal donors the maximal numbers of oocytes are available at 4 weeks of age, when ovaries bear peak numbers of antral follicles. Thus, 4 weeks old lambs are considered good donors, even if the developmental competence of prepubertal oocytes is lower compared to their adult counterpart.

Basic research and commercial applications would be boosted by the possibility of successfully cryopreserving vitrified oocytes obtained from both adult and prepubertal donors. The vitrification of oocyte collected from prepubertal donors would also allow shortening the generation interval and thus increasing the genetic gain in breeding programs. However, the loss of developmental potential after cryopreservation makes mammalian oocytes probably one of the most difficult cell types to cryopreserve. Among the available cryopreservation techniques, vitrification is widely applied to animal and human oocytes. Despite recent advancements in the technique, exposures to high concentrations of cryoprotective agents as well as chilling injury and osmotic stress still induce several structural and molecular alterations and reduce the developmental potential of mammalian oocytes. Here, we describe a protocol for the vitrification of sheep oocytes collected from juvenile and adult donors and matured in vitro prior to cryopreservation. The protocol includes all the procedures from oocyte in vitro maturation to vitrification, warming and post-warming incubation period. Oocytes vitrified at the MII stage can indeed be fertilized following warming, but they need extra time prior to fertilization to restore damage due to cryopreservation procedures and to increase their developmental potential. Thus, post-warming culture conditions and timing are crucial steps for the restoration of oocyte developmental potential, especially when oocyte are collected from juvenile donors.

Introduction

Long-term storage of the female gametes can offer a wide range of applications, such as improving domestic animal breeding by genetic selection programs, contributing to preserve biodiversity through the ex-situ wildlife species conservation program, and boosting in vitro biotechnology research and applications thanks to the availability of stored oocytes to be incorporated in in vitro embryo production or nuclear transplantation programs^1,2,3. Juvenile oocyte vitrification would also increase genetic gain by shortening the generation interval in breeding programs⁴. Vitrification by ultra-rapid cooling and warming of oocytes is currently considered a standard approach for livestock oocytes cryopreservation⁵. In ruminants, before vitrification, oocytes are usually matured in vitro, after retrieval from follicles obtained from abattoir-derived ovaries². Adult, and especially prepubertal ovaries^4,6, can indeed supply a virtually unlimited number of oocytes to be cryopreserved.

In cattle, after oocyte vitrification and warming, blastocyst yields at >10% have been commonly reported by several laboratories during the last decade³. However, in small ruminants oocyte vitrification is still considered relatively new for both juvenile and adult oocytes, and a standard method for sheep oocyte vitrification remains to be established^2,5. Despite recent advancements, the vitrified and warmed oocyte indeed presents several functional and structural alterations that limit their developmental potential^7,8,9. Thus, few articles have reported blastocyst development at 10% or more in vitrified/warmed sheep oocytes². Several approaches have been investigated to reduce the above-mentioned alterations: optimizing the composition of the vitrification and thawing solutions^10,11; experimenting with the use of different cryo-devices^8,12,13; and applying specific treatments during in vitro maturation (IVM)^4,14,15 and/or during the recovery time after warming⁶.

Here we describe a protocol for the vitrification of sheep oocytes collected from juvenile and adult donors and matured in vitro prior to cryopreservation. The protocol includes all the procedures from oocyte in vitro maturation to vitrification, warming and post-warming culture period.

Protocol

The animal protocol and the implemented procedures described below are in accordance with the ethical guidelines in force at the University of Sassari, in compliance with the European Union Directive 86/609/EC and the recommendation of the Commission of the European Communities 2007/526/EC.

1. Preparation of media for oocyte manipulation

1. Prepare the medium for transport of collected ovaries by supplementing Dulbecco's phosphate buffered saline with 0.1 g/L penicillin and 0.1 g/L streptomycin (PBS).
2. Prepare the medium for oocyte collection and maturation by diluting 9.5 g of Tissue Culture Medium (TCM) 199 in powder with 1 L of Milli-Q water supplemented with penicillin (0.1%) and streptomycin (0.1%).
   1. After dilution, filter 100 mL of medium and store it at 4 °C as Stock Maturation Medium (SMM) to be used for one week.
   2. Prepare the collection medium (CM) by supplementing the remaining 900 mL with 25 mM HEPES, 0.36 g/L bicarbonate and 0.1% (w/v) polyvinyl alcohol (PVA) (pH 7.3, osmolality 290 mOsm/kg).
3. Prepare the maturation medium with SMM supplemented with 0.021 g/L bicarbonate, 10% heat-treated estrus sheep serum, 1 IU/mL FSH, 1 IU/mL LH, 100 µL of cysteamine and 8 mg/mL of pyruvate.
   NOTE: The maturation medium in a volume of 10 mL must be incubated at standard conditions (in a maximum humidified atmosphere at 39 °C in 5% CO₂ in air) for at least 4 h before use.
4. Prepare the base medium (BM) for manipulation of oocyte after in vitro maturation, consisting in PBS without Ca⁺⁺ and Mg⁺⁺, supplemented with 20% fetal calf serum (FCS).

2. Oocyte collection and maturation

1. Recover the oocytes from juvenile (30-40 days of age, body weight 6-10 kg) and adult ovaries.
2. Transport the collected ovaries from the commercial slaughterhouse to the laboratory within 1-2 h in PBS at 27 °C.
3. After washing in PBS fresh medium, slice the ovaries in CM using a micro-blade to release the follicle content.
4. Under a stereomicroscope examination with 60x magnification, select cumulus-oocyte complexes (COCs) for in vitro maturation by choosing those with 4-10 layers of granulosa cells, oocyte with a uniform cytoplasm, homogeneous distribution of lipid droplets in the cytoplasm and with the outer diameter of about 90 µm (mean).
5. Wash the selected COCs three times in CM and finally transfer them in maturation medium.
   NOTE: For juvenile oocytes, to improve survival after vitrification, supplement the maturation medium with 100 µM trehalose.
6. For in vitro maturation, transfer 30-35 COCs in 600 µL of maturation medium in four-well Petri dishes, covered with 300 µL of mineral oil and incubate them for 22 (adult oocytes)/24 (juvenile oocytes) h in 5% CO₂ in air at 39 °C.
7. After in vitro maturation, denude COCs of cumulus cells by gently pipetting. Following the examination under a stereomicroscope with 60x magnification, select only those showing the extrusion on the first polar body, and thus at metaphase II (MII) stage, for vitrification.

3. Semen collection, freezing and thawing procedures

1. Prepare the base medium for semen cryopreservation consisting in ram extender (200 mM Tris; 70 mM citric acid; 55 mM fructose; pH 7.2, osmolality 300 mOsm/kg) supplemented with egg yolk 20% (v/v).
2. Collect the semen only during sheep breeding season (October-November).
3. Obtain ejaculates by artificial vagina from adult rams (aged 2-5 years), maintained in an outdoor environment and fed a live-weight maintenance ration. Keep rams isolated in separate pens, but with visual contact between each other.
4. Repeat semen collection one a week during the entire breeding season to obtain at least 8 ejaculates from each male.
5. Transport the semen samples to the laboratory at environmental temperature within 5 min after collection and immediately process. Pool the ejaculates of two-three rams and evaluate sperm concentration spectrophotometry.
6. After pooling, dilute the ejaculates up to 400 x 10⁶ spermatozoa/mL with base medium for semen cryopreservation supplemented with 4% glycerol. Then cool the diluted semen to 4 °C over a period of 2 h and equilibrate it for 20 min before freezing.
7. Freeze the semen samples in pellet form (0.25 mL) on dry ice and then plunge them into liquid nitrogen.
8. For thawing, put the pellet in a sterilized glass falcon and plunge it in a water bath for 20 s at 39 °C.

4. In vitro fertilization and embryo culture

1. Prepare stocks for constitution of the synthetical oviductal fluid (SOF).
   1. Prepare Stock A: 99.4 mL of MilliQ-water; 6.29 g of NaCl; 0.534 g of KCl; 0.161 g of KH₂PO₄; 0.182 g of MgSO₄ ·7H₂O; and 0.6 mL of Sodium Lactate. Keep at 4 °C for up to 3 months.
   2. Prepare Stock B: 10 mL of MilliQ-water; 0.210 g of NaHCO₃; and 2-3 g of Phenol Red. Keep at 4 °C for 1 month.
   3. Prepare Stock C: 10 mL of MilliQ-water; and 0.051 g of sodium pyruvate. Keep at 4 °C for 1 month.
   4. Prepare Stock D: 10 mL of MilliQ-water; and 0.262 g of CaCl₂ 2H₂O. Keep at 4 °C for 1 month.
   5. Prepare 10 mL of SOF consisting of 7.630 mL of MilliQ water, 1 mL of Stock A, 1 mL of Stock B, 0.07 mL of Stock C and 0.7 mL of stock D.
   6. Prepare in vitro fertilization (IVF) medium: SOF supplemented with 2% heat-treated estrous sheep serum, 10 µg/mL heparin and 1 µg/mL hypoutarine (osmolality 280-290 mOsm/kg).
      NOTE: The IVF medium in a volume of 10 mL must be incubated at standard conditions (in a maximum humidified atmosphere at 39 °C in 5% CO₂, 5% O₂ and 90% N₂) at least 4 h before use.
2. Transfer frozen/thawed semen aliquots in a sterilized glass conical tube below 1.5 mL of warmed IVF medium and incubate them for 15 min at 39 °C in a humidified atmosphere at 5% CO₂ in air.
3. After incubation, the motile spermatozoa swim towards the apical portion of the liquid column. Collect the top layer and observe for sperm motility evaluation.
   NOTE: Sperm motility parameters should be assessed using a computer-assisted sperm analysis (CASA) system with the following settings: 25 frames acquired to avoid sperm track overlapping, minimum contrast 10, minimum velocity of average path 30 µm/s, and progressive motility > 80% straightness. This system has a specific setup for ram sperm evaluation. For each sample, 5 µL subsample of sperm suspension are loaded into a pre-warmed analysis chamber with a depth of 10 µm. Sperm motility is assessed at 37 °C at 40x using a phase contrast microscope and a minimum of 500 sperm per subsample should be analyzed in at least four different microscopic fields. The percentage of total motile and progressive motile sperm were evaluated. For the IVF, the percentage of progressive motile spermatozoa should be ≥ 30%.
4. Dilute swim-up derived motile spermatozoa at 1 x 10⁶ spermatozoa/mL final concentration and co-incubate them with MII oocytes in 300 µL of IVF medium covered with mineral oil in four-well Petri dishes.
5. After 22 h transfer the presumptive zygotes in four-well Petri dishes containing SOF supplemented with 0.4% bovine serum albumin and essential and non-essential amino acids at oviductal concentration as reported by¹⁶ under mineral oil and culture them under standard conditions up to the blastocyst stage.
6. At 22-, 26- and 32- h post-insemination, record the number of cleaved oocytes, showing two distinct blastomeres, by the examination under a stereomicroscope with 60x magnification.
7. Observe the embryos daily starting from the fifth to the ninth day of culture and newly formed blastocysts should be recorded by the examination under a stereomicroscope with 60x magnification.

5. Oocyte vitrification and warming

NOTE: Perform vitrification following the method of minimum essential volume (MEV) using device cryotops¹⁷.

1. Equilibrate a group of five oocytes at 38.5 °C for 2 min in BM. The use of BM guarantees a low calcium concentration ([Ca²⁺⁺] 2.2 mg/dL)¹⁰.
2. Dehydrate the oocytes with a 3 min exposure to equilibration solution containing 7.5% (v/v) dimethyl sulfoxide (DMSO) and 7.5% (v/v) ethylene glycol (EG) in BM.
3. Transfer the oocytes to the vitrification solution containing 16.5% (v/v) DMSO, 16.5% (v/v) EG and 0.5 M trehalose in BM before loading them in a cryotop device and directly plunging them into liquid nitrogen within 30 s.
4. To warm to a biological temperature, transfer the content of each vitrification device from liquid nitrogen into 200 µL drops of 1.25 M trehalose in BM for 1 min at 38.5 °C, and gently stir to facilitate the mixing.
5. To promote removal of intracellular cryoprotectants, transfer oocytes stepwise into 200 µL drops of decreasing trehalose solutions (0.5 M, 0.25 M, 0.125 M trehalose in BM) for 30 s at 38.5 °C before being equilibrated for 10 min at 38.5 °C in BM.

6. Assessment of oocyte quality post-warming

1. After warming, incubate the oocytes for 6 h in PBS without Ca⁺⁺ and Mg⁺⁺ plus 20% FCS (BM) in 5% CO₂ in air at 38.5 °C.
   NOTE: The oocyte ability to restore biological and structural features after vitrification is in relation to the species and classes of used oocytes.
2. Since the oocyte ability to recover cryopreservation damages is time-dependant, assess oocyte quality at different time points of in vitro culture (0 h, 2 h, 4 h, 6 h) after warming, to define the optimal time window for oocyte fertilization.
   ​NOTE: In adult sheep oocyte, the optimal time is 4 h post-warming; for prepubertal oocyte, the optimal time is 2 h post-warming.

7. Oocyte survival assessment

1. Immediately after warming and for each time point of post-warming culture, morphologically evaluate oocytes using an inverted microscope with 100x magnification.
   NOTE: Oocytes with structural alterations, such as faint cytoplasm, damage zona pellucida and/or membrane should be classified as degenerated.
2. Validate the membrane integrity evaluation using a double differential fluorescent staining.
3. Incubate the oocytes in 2 mL of BM containing propidium iodide (PI; 10 µg/mL) and Hoechst 33342 (10 µg/mL) for 5 min in 5% CO₂ in air at 38.5 °C.
4. After washing three times in fresh BM, observe the oocytes under a fluorescent microscope using an excitation filter of 350 nm and emission of 460 nm for Hoechst 33342 and an excitation filter of 535 nm and emission of 617 nm for PI.
   ​NOTE: Oocytes with an intact membrane can be recognized by the blue fluorescence of colored DNA with Hoechst 33342. Oocyte with damaged membranes show a red fluorescence due to DNA staining with PI.

8. Evaluation of mitochondrial activity and ROS intracellular levels by confocal laser scanning microscopy

1. Prepare the MitoTracker Red CM-H₂XRos (MT-Red) probe.
   1. Dilute the content of 1 vial (50 µg) with 1 mL of DMSO to obtain a 1 mM solution. Keep the diluted vial in liquid nitrogen.
   2. Dilute the solution 1 mM with DMSO to obtain the 100 µM stock solution and store it at -80 °C in the dark.
2. Prepare 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA) probe.
   1. Dilute the H₂DCF-DA in 0.1% polyvinyl pyrrolidone (PVA)/PBS to obtain the first 100 mM solution. Keep the solution at -80 °C in the dark .
   2. Dilute the first solution in 0.1% PVA/PBS to obtain the 100 µM stock solution. Store it at -20 °C in the dark.
3. Prepare the mounting medium (MM): for 10 mL of MM, add 5 mL of glycerol, 5 mL of PBS and 250 mg of sodium azide. Store it at -20 °C until use.
4. Incubate the oocytes for 30 min at 38.5 °C in BM with 500 nM MT-Red (stock solution: 100 µM in DMSO).
5. After incubation with MT-Red probe, wash the oocytes three times in BM and incubate for 20 min in the same media containing 5 µM H₂DCF-DA (stock solution: 100 µM in BM).
6. After exposure to the probes, wash the oocytes in BM and fix in 2.5% glutaraldehyde/PBS for at least 15 min.
7. After fixation, wash the oocytes three times in BM and mount on glass slides in a 4 µL drop of MM with 1 mg/mL Hoechst 33342 using wax cushions to avoid compression of samples.
8. Store slides at 4 °C in the dark until evaluation.
9. Perform the analysis of immunolabelled sections with a confocal laser scanning microscope. The microscope is equipped with Ar/He/Ne lasers, using a 40/60x oil objective. Analyze the sections by sequential excitation.
10. For mitochondrial evaluation, observe the samples with a multiphoton laser to detect MT-Red (exposure: 579 nm; emission: 599 nm).
11. Use an argon ions laser ray at 488 nm and the B-2 A filter (495 nm exposure and 519 nm emission) to point out the dichlorofluorescein (DCF)¹⁸.
12. In each individual oocyte, measure MT-Red and DCF fluorescence intensities at the equatorial plane¹⁹.
13. Maintain parameters related to fluorescence intensity at constant values during all image acquisitions (laser energy 26%, Sequential Settings 1: PMT1 gain 649-PMT2 gain 482; Sequential Setting 2: PMT1 gain 625-PMT2 gain 589; offset 0; pinhole size: 68).
14. Perform quantitative analysis of fluorescence intensity using the Leica LAS AF Lite image analysis software package, following the procedures standardized by²⁰.
15. Capture the pictures once, moving on the Z axis, until reaching the equatorial plane.
16. For each photo, transform to gray scale and turn off channel 1 (related to Hoechst blue) was turned off. Then manually draw a region of interest (ROI) on a circumscribed area, that is around the meiotic spindle.
    NOTE: The software can automatically read the pixel average value on the channel 2 (FITC), subtracting the value of the background from it.
17. Record the mean values of pixels and submit for statistical analysis.

9. Statistical Analyses

1. Analyze the following differences: survival rates in juvenile vs adult oocytes, survival rates and developmental competence in control and trehalose-treated juvenile oocytes, survival and parthenogenetic activation rates and developmental competence of adult oocytes vitrified with different calcium concentration media, fertilization rates and embryo production in juvenile oocytes vitrified with low or high calcium concentration, active mitochondria phenotypes in juvenile vitrified oocytes during different time points of post-warming culture and parthenogenetic activation rates between adult and juvenile vitrified oocytes using the chi square test.
2. Analyze the cleavage rate and embryo output in vitrified adult oocytes during different time points of post-warming culture, fluorescence intensity of mitochondrial activity and ROS intracellular levels in juvenile vitrified oocytes during different time points of post-warming culture by ANOVA after analysis for homogeneity of variance by Levene's test. Use a post-hoc test Tukey's test to highlight differences between and among groups.
3. Perform statistical analysis using the statistical software program and consider a probability of P < 0.05 to be the minimum level of significance. All results are expressed as mean ± S.E.M.

Representative Results

The cryotolerance of oocyte from juvenile donors is lower compared to adult ones. The first effect observed is a lower post-warming survival rate compared to adult oocytes (Figure 1A; χ² test P<0.001). Juvenile oocytes showed a lower membrane integrity after warming (Figure 1B). The use of trehalose in the maturation medium was intended to verify whether this sugar could reduce cryoinjuries in juvenile oocytes. The data have demonstrated²³ that oocytes matured for 24 h with trehalose supplementation showed higher survival rates after vitrification/warming compared to the non-treated group (Table 1: 85.7% vs 75.3% respectively; χ² test P<0.05). Trehalose supplementation was indeed associated with higher membrane integrity after warming (Figure 2A). Thus, the use of trehalose during the in vitro maturation of juvenile oocytes increased the rates of survival after vitrification (85.7%) to values comparable to adult ones (90.3%). However, cleavage, fertilization and developmental rates of juvenile oocytes were not increased by trehalose supplementation (Table 1).

To improve oocyte competence after vitrification we tested in adult ovine oocytes different vitrification media with calcium concentrations ranging from 9.9 to 0.4 mg/dL¹⁰. Obtained results showed that the use of media with calcium concentration equal to 2.2 mg/dL increased post-warming survival rates, improved developmental competence and reduced parthenogenetic activation of adult oocyte¹⁰ (Table 2). We thus tested the low calcium vitrification media for the vitrification of juvenile oocytes. As shown in Table 3, juvenile oocytes vitrified with low calcium concentration evidenced higher fertilization rates compared to oocytes vitrified with high calcium concentration (44.35% vs 32.29 % respectively; P<0.05), but no differences were found in embryo production.

Vitrified/warmed oocytes need extra time prior to fertilization to restore damage due to cryopreservation procedures and to increase their developmental potential. A previous study has indeed demonstrated that ATP intracellular concentration, mitochondrial activity and in vitro developmental competence are reduced in vitrified/thawed oocytes, which also show high intracellular ROS concentrations⁶. These alterations are particularly marked immediately after warming. During the post-warming culture, both adult and juvenile oocytes are able to partially recover from the damages suffered during the vitrification procedures^{6, 24}. By comparing post-warming culture of different durations (0, 2, 4, and 6 h), we showed that after 4 h of culture oocytes collected from adult ewes are able to recover the energetic balance⁶ and microtubular setup²⁴ and to restore the developmental competence with higher cleavage (50.7 ± 3.9%; P< 0.01 ANOVA) and blastocyst rates (14.40 ± 1.3%; ANOVA P< 0.01) compared to other time points (0, 2 and 6 h; Table 4). Thus, 4 h of post-warming culture represents the ideal time window for fertilization of vitrified/warmed adult oocytes⁶.

When the same experiment was repeated with oocytes collected from juvenile donors, these results were partially confirmed. Mitochondrial activity was higher in vitrified/warmed juvenile oocytes after 4 h of post-warming culture compared to other time points (0, 2, 6; Figure 3: ANOVA P<0.01). Several patterns of mitochondrial distribution are observed and classified into the following three groups (as reported by²¹ ): Pattern A: homogeneous FINE with small granulations spread throughout the cytoplasm; Pattern B: homogeneous GRANULAR with large granulations spread throughout the cytoplasm; Pattern C: heterogeneous CLUSTERED when particularly large granulations were present, spread all over the cytoplasm or located in specific cytoplasmic domains. The different phenotypes in the cytoplasm distribution of active mitochondrial in MII can be related to oocyte developmental competence. A FINE homogeneous distribution is an indicator of poor developmental competence while a GRANULAR and CLUSTERED distribution are related to an increased mitochondria activity and consequently higher developmental competence²². Mitochondrial distribution patterns changed during 6 h of post-warming culture. Figure 4 shows examples of juvenile oocytes having different patterns of mitochondrial distribution and their fluctuations during post-warming culture. The pattern A increase significantly during the prolonged incubation and reaches the higher value at 6 h post-warming (Figure 4Aa: χ² P<0.05), the pattern B did not show significant changes during post-warming culture (Figure 4Bb), the pattern C was not found in any juvenile vitrified/warmed oocyte during the prolonged incubation (Figure 4Cc: χ² P<0.05).

Moreover, ROS intracellular levels were significantly lower in juvenile oocytes at 2 h of post-warming culture compared to 0, 4 and 6 h (Figure 5: ANOVA P<0.001). However, and in contrast to what was found in adult oocytes, the rate of spontaneous parthenogenetic activation increased during the post-warming culture in juvenile oocytes (Figure 6). For this reason, the recommended time point for fertilization in juvenile oocytes would be 2 h after the post warming culture.

Figure 1. Survival rates of vitrified/warmed ovine oocyte collected from juvenile and adult donors. (A) Oocyte were vitrified after in vitro maturation. Survival rates were determined after vitrification and warming by fluorescent staining with propidium iodide (10 µg/mL) and Hoechst 33342 (10 µg/mL). N = 165 adult oocytes and 170 juvenile oocytes. Different letters indicate significant differences between adult and juvenile oocytes: χ² test P<0.001. (B-C) Examples of vitrified/warmed juvenile oocytes with intact (B) and damaged plasma membrane (C) at the morphological evaluation (inverted microscope with 100x magnification). Scale bar = 10 µm. Please click here to view a larger version of this figure.

Figure 2. Juvenile ovine oocyte survival rates and in vitro developmental competence after maturation with (TRH) and without (CTR) trehalose (100 mM in maturation medium). (A-B) Examples of juvenile oocytes vitrified after in vitro maturation in media supplemented (A) with or (B) without trehalose at the morphological evaluation (inverted microscope with 100x magnification). Scale bar = 30 µm. Please click here to view a larger version of this figure.

Figure 3. Quantification of active mitochondrial fluorescence intensity in vitrified/warmed juvenile oocytes at different time points (0, 2, 4, 6) during post-warming in vitro culture. IVM oocytes were used as a control (CTR N = 77). In total 163 (0 h N = 45; 2 h N = 39; 4 h N = 40; 6 h N = 39) juvenile oocytes were vitrified and warmed in three independent experiments. Different letters indicate statistically significant differences (ANOVA P = 0.0000). Please click here to view a larger version of this figure.

Figure 4. Distribution of mitochondrial pattern in vitrified/warmed juvenile oocytes during 6 hours of post-warming in vitro culture. Representative images of Fine (A), Granular (B) and Clustered (C) mitochondrial distribution in vitrified/warmed juvenile oocytes. (D) Percentage of juvenile vitrified/warmed oocytes showing a fine mitochondrial distribution; (E) Percentage of juvenile vitrified/warmed oocytes showing a granular mitochondrial distribution; (F) Percentage of juvenile vitrified/warmed oocytes showing a clustered mitochondrial distribution. IVM juvenile oocytes were used as control (CTR; N = 77). In total 163 (0 h N = 45; 2 h N = 39; 4 h N = 40; 6 h N = 39) juvenile oocytes vitrified and warmed in three independent experiments were used. Different letters indicate statistically significant differences (Aa: χ² P = 0.026; Bb: χ² P = 0.097; Cc: χ² P = 0.014). Scale bar = 30 µm. Please click here to view a larger version of this figure.

Figure 5. Quantification of intracellular ROS fluorescence intensity in vitrified juvenile oocytes during of 6 hours post-warming in vitro culture. (A-B) Representative images of ROS fluorescence intensity in in vitro matured (A) and vitrified (B) juvenile oocytes. (C) Intracellular levels of ROS as determined by quantification of fluorescence intensity in vitrified juvenile oocytes at different time points (0 h N=45; 2 h N=39; 4 h N=40; 6 h N=39) during post-warming in vitro culture. In vitro matured juvenile oocytes were used as a control (CTR N = 77). Different letters indicate statistically significant differences (ANOVA P = 0.0000). Scale bar = 50 µm. Please click here to view a larger version of this figure.

Figure 6. Parthenogenetic activation in vitrified juvenile and adult oocytes during 6 hours of post-warming in vitro culture. (A-B) Representative images of oocyte parthenogenetic activation: (A) oocyte in metaphase II-telophase II transition and (B) pronucleus formation. (C) Percentages of parthenogenetic activated adult and juvenile oocytes at different time points (0, 2, 4, 6 h) during post-warming in vitro culture. Asterisks indicate statistical differences between juvenile and adult oocytes at each time point of incubation (ANOVA P = 0.000). This figure has been modified from Serra et al.²⁴ Scale bar = 50 µm. Please click here to view a larger version of this figure.

	Oocytes (n)	Survival rate (%)	IVF (n)	Fertilizeda (%)	Cleavedb (%)	Blastocystsc (%)
CTR	73	75.3*	452	91.1	96.1	14.3
TRH	77	85.7	470	92.5	95.4	13
* Chi square test p<0.5
a Percentages are calculated on IVF oocytes
b Percentages are calculated on fertilized oocytes
c Percentages are calculated on cleaved oocytes

Table 1. Juvenile ovine oocytes survival rates and in vitro developmental competence after maturation with and without trehalose and vitrification. TRH = juvenile oocytes matured with trehalose supplementation (100 mM in maturation medium). CTR = control juvenile oocytes matured without trehalose supplementation. Survival rates were determined after fluorescent staining with propidium iodide (10 µg/mL) and Hoechst 33342 (10 µg/mL) of vitrified/warmed oocytes. Oocyte developmental competence was determined after incorporation in an in vitro production system. ^a Percentages are calculated on IVF oocytes. ^b Percentages are calculated on fertilized oocytes. ^c Percentages are calculated on cleaved oocytes. * χ² test P<0.5. This table has been modified from Berlinguer et al.²³

Groups	[Ca++] mg/dL	N oocyte	Spontaneous parthenogenetic activation (%)	Vitrified oocytes	Survived and IVF oocytes (%)	Cleavage (%)	Blastocysts output (%)
TCM/FCS	9.9	80	33 (41.2)a	150	124 (82.7)a	40 (32.5)a	2 (1.6)a
PBS/FCS	4.4	82	29 (35.3)ac	115	88 (76.5)a	33 (37.5)ae	1 (1.1)a
PBSCaMg free/FCS	2.2	86	11 (12.7)b	126	115 (91.3)b	74 (64.3)b	12 (10.4)b
PBS/BSA	3.2	83	21 (25.3)c	110	90 (81.8)a	18 (20)c	0 (0)a
PBSCaMg free/BSA	0.4	87	10 (11.5)b	149	123 (82.5)a	57 (46.3)de	3 (2.4)b

Table 2. Developmental competence of in vitro matured adult oocytes vitrified in vitrification media (16.5% ethylene glycol + 16.5% dimethyl sulfoxide) containing different calcium concentrations. Survival and fertilization rates are calculated on vitrified oocytes; total cleavage and blastocyst rates are calculated on survived oocytes. Values with different subscript within the same column are significantly different: χ² test P<0.05. This table has been modified from Succu et al.¹⁰

[Ca 2++] in vitrification media	No. Oocytes	Post-vitrification survival rate (%)	Fertilization rate (%)	Cleavage (%)	Blastocyst output (%)
High [9.9 mg/dL]	190	161 (84.73)	52/161 (32.29)a	43/161 (26.7)	0
Low [2.2 mg/dL]	150	124 (82.66)	55/124 (44.35)b	41/124 (33.1)	0

Table 3. Fertilization and developmental rates after in vitro fertilization and culture of vitrified/warmed juvenile oocyte using high ([Ca 2++] = 9.9 mg/dL) and low ([Ca 2++] = 2.2 mg/dL) calcium concentration in vitrification media. Different letters indicate statistical difference (a ≠ b P<0.05 χ² test).

Hours of post-warming incubation	Cleavage rate (n)	Embryo output (n)
0	19.2 ± 3%a (82)	0%a (17)
2	41.8 ± 3%b (100)	6.5 ± 1.3%b (42)
4	50.7 ± 3%b (92)	14.4 ± 1.3%c (48)
6	26 ± 3%a (92)	0%a (23)

Table 4. Cleavage rate and embryo output in vitrified/warmed adult oocytes fertilized at different time points of post-warming culture. Different letters indicate statistical difference within the same column: ANOVA P<0.01.

Discussion

Oocyte cryopreservation in domestic animals can allow not only the long-term conservation of female genetic resources, but also advance the development of embryonic biotechnologies. Thus, the development of a standard method for oocyte vitrification would advantage both the livestock and the research sector. In this protocol, a complete method for adult sheep oocyte vitrification is presented and could represent a solid starting point for the development of an efficient vitrification system for juvenile oocyte.

One of the main advantages of the proposed method is that it includes all the steps from oocyte collection, in vitro maturation, vitrification, and warming. Moreover, it includes a post-warming culture period to allow oocytes to recover from the damages incurred during the vitrification procedure before being fertilized. The optimum time for fertilization should be tailored according to the method of cryopreservation, initial oocyte quality, patient age, and species, being essential to consider both aspects of time recovery and oocyte aging^25,26. Thus, choosing the duration of the post-warming incubation period is challenging and it may impact the outcome of oocyte vitrification programs. Based on the results obtained in terms of cleavage rates and embryo output, and under the conditions described in the presented protocol, the optimum time for fertilization of vitrified adult sheep oocytes is after 4 hours of post-warming incubation (Table 4)⁶. This information is crucial when designing an oocyte vitrification program.

This protocol, however, while giving acceptable results in terms of embryo output from vitrified/warmed adult oocytes, still leads to low to zero embryos if applied to juvenile oocytes. Several structural and functional limitations impair prepubertal oocyte developmental competence, such as small size, defective coupling between cumulus cells and oocytes, decrease in amino acid uptake, reduced protein synthesis and energy metabolism ^27,28,29. In a previous study we reported that prepubertal oocytes show high sensitivity to the vitrification procedure³⁰. The low developmental competence shown after vitrification and warming is probably the result of damages to cytoplasmic factors involved in the reorganization of the cytoskeleton and (or) in the activation of maturation promoting factor³⁰. As shown in Table 1, the supplementation of the maturation medium with trehalose, a non-permeable cryoprotectant, was able to increase survival rates after vitrification and warming to values comparable to those of adult oocytes⁴. In the same way, the use of vitrification solution with low calcium concentrations increases fertilization rates of juvenile oocytes after vitrification and warming, as shown in Table 3. Thus, both the optimization on culture conditions during in vitro maturation and of the vitrification media composition may help in increasing the quality of the juvenile oocyte after vitrification and warming. Juvenile oocytes show some ability to recover from the damages induced by the vitrification procedure, as shown in Figure 3, 4 and 5.

However, the high rates of spontaneous parthenogenetic activation during post-warming culture still limit their developmental potential. Ethylene glycol and DMSO, which are commonly used cryoprotective agents, may artificially activate the oocyte before the actual fertilization, thereby limiting fertilization success and embryo development. They can indeed cause a transient increase in intracellular Ca²⁺ concentration³¹, thus triggering cortical granule exocytosis, pronuclei formation, and meiotic resumption³². In fact, vitrification may artificially activate the oocyte before the actual fertilization, thereby limiting fertilization success and embryo development. Calcium chelator may thus be used to further limit calcium availability during the vitrification process with the aim of limiting the rate of spontaneous activation in juvenile oocytes.

It should also be considered that, unlike slow-freezing, vitrification is an exclusively manual technique and it is thus operator dependent^33,34. Thus, the availability of trained personnel is a key factor for the success of this method. First of all, the operator has to properly select the oocytes to be vitrified. After IVM, oocytes are gently denuded of cumulus cells and evaluated under a stereomicroscope to select for cryopreservation only those with a uniform cytoplasm, homogeneous distribution of lipid droplets in the cytoplasm and with the outer diameter of about 90 µm. Moreover, only oocytes showing the extrusion on the first polar body, and thus at the MII stage, must be selected.

The morphological evaluation of the oocytes must be completed in a few minutes and being operator-dependant, it is very sensitive to variations in its proper implementation. To help standardize the selection procedure, the method suggests limiting the culture time for in vitro maturation to 22 h for adult oocytes. At this time point, sheep oocytes of high quality have already completed the first meiotic division²² and can be selected for cryopreservation. This way the elimination of low-quality oocytes, which are the slowest ones in the completion of the first meiotic division, should be simpler.

The operator must also strictly respect the timing set for the vitrification procedure, from the first exposure to the cryoprotectant to the immersion in liquid nitrogen. Another critical step is the loading of the oocyte in the vitrification device used. The procedure must use minimum sample volumes to increase the cooling rate and to help cells pass through the phase transition temperature rapidly, thereby decreasing cryoinjuries³⁵. Cryotop uses a polypropylene strip attached to a holder. In this method, the oocytes in the vitrification solution (<0.1 µL) are rapidly loaded with a glass capillary on top of the film strip. Then, the solution must be removed, leaving behind a thin layer sufficient to cover the cells to be cryopreserved. Once again, this step must be completed as fast as possible to limit the exposure of the oocytes to the high cryoprotectant concentrations of the vitrification solution, which can cause osmotic shock and are toxic to the cells.

For these reasons, a major challenge associated with this method is the need for manual handling and skilled technician. Other authors reported that the oocyte vitrification outcome appears influenced by a "learning curve" effect, as the acquisition of manual skills can significantly reduce the biological damage induce by the vitrification procedure³⁴. Researchers should thus take into consideration the "operator effect" in the evaluation of the outcome of the vitrification procedure.

Further studies will focus both on standardizing the oocyte selection procedure and better tailoring media composition and culture conditions to the needs of the juvenile oocytes. At this regard, both the use of calcium chelator and antioxidants may offer promising opportunities. Similarly, the optimization of the protocol will allow increasing the developmental competence of vitrified/warmed adult oocytes.

Materials

Name	Company	Catalog Number	Comments
2′,7′-Dichlorofluorescin diacetate	Sigma-Aldrich	D-6883
Albumin bovine fraction V, protease free	Sigma-Aldrich	A3059
Bisbenzimide H 33342 trihydrochloride (Hoechst 33342)	Sigma-Aldrich	14533
Calcium chloride (CaCl2 2H20)	Sigma-Aldrich	C8106
Citric acid	Sigma-Aldrich	C2404
Confocal laser scanning microscope	Leica Microsystems GmbH,Wetzlar	TCS SP5 DMI 6000CS
Cryotop Kitazato	Medical Biological Technologies
Cysteamine	Sigma-Aldrich	M9768
D- (-) Fructose	Sigma-Aldrich	F0127
D(+)Trehalose dehydrate	Sigma-Aldrich	T0167
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D2438
Dulbecco Phosphate Buffered Saline	Sigma-Aldrich	D8537
Egg yolk	Sigma-Aldrich	P3556
Ethylene glycol (EG)	Sigma-Aldrich	324558
FSH	Sigma-Aldrich	F4021
Glutamic Acid	Sigma-Aldrich	G5638
Glutaraldehyde	Sigma-Aldrich	G5882
Glycerol	Sigma-Aldrich	G5516
Glycine	Sigma-Aldrich	G8790
Heparin	Sigma-Aldrich	H4149
HEPES	Sigma-Aldrich	H4034
Hypoutarine	Sigma-Aldrich	H1384
Inverted microscope	Diaphot, Nikon
L-Alanine	Sigma-Aldrich	A3534
L-Arginine	Sigma-Aldrich	A3784
L-Asparagine	Sigma-Aldrich	A4284
L-Aspartic Acid	Sigma-Aldrich	A4534
L-Cysteine	Sigma-Aldrich	C7352
L-Cystine	Sigma-Aldrich	C8786
L-Glutamine	Sigma-Aldrich	G3126
LH	Sigma-Aldrich	L6420
L-Histidine	Sigma-Aldrich	H9511
L-Isoleucine	Sigma-Aldrich	I7383
L-Leucine	Sigma-Aldrich	L1512
L-Lysine	Sigma-Aldrich	L1137
L-Methionine	Sigma-Aldrich	M2893
L-Ornithine	Sigma-Aldrich	O6503
L-Phenylalanine	Sigma-Aldrich	P5030
L-Proline	Sigma-Aldrich	P4655
L-Serine	Sigma-Aldrich	S5511
L-Tyrosine	Sigma-Aldrich	T1020
L-Valine	Sigma-Aldrich	V6504
Magnesium chloride heptahydrate (MgSO4.7H2O)	Sigma-Aldrich	M2393
Makler Counting Chamber	Sefi-Medical Instruments ltd.Biosigma S.r.l.
Medium 199	Sigma-Aldrich	M5017
Mineral oil	Sigma-Aldrich	M8410
MitoTracker Red CM-H2XRos	ThermoFisher	M7512
New born calf serum heat inactivated (FCS)	Sigma-Aldrich	N4762
Penicillin G sodium salt	Sigma-Aldrich	P3032
Phenol Red	Sigma-Aldrich	P3532
Polyvinyl alcohol (87-90% hydrolyzed, average mol wt 30,000-70,000)	Sigma-Aldrich	P8136
Potassium Chloride (KCl)	Sigma-Aldrich	P5405
Potassium phosphate monobasic (KH2PO4)	Sigma-Aldrich	P5655
Propidium iodide	Sigma-Aldrich	P4170
Sheep serum	Sigma-Aldrich	S2263
Sodium azide	Sigma-Aldrich	S2202
Sodium bicarbonate (NaHCO3)	Sigma-Aldrich	S5761
Sodium chloride (NaCl)	Sigma-Aldrich	S9888
Sodium dl-lactate solution syrup	Sigma-Aldrich	L4263
Sodium pyruvate	Sigma-Aldrich	P2256
Sperm Class Analyzer	Microptic S.L.	S.C.A. v 3.2.0
Statistical software Minitab 18.1	2017 Minitab
Stereo microscope	Olimpus	SZ61
Streptomycin sulfate	Sigma-Aldrich	S9137
Taurine	Sigma-Aldrich	T7146
TRIS	Sigma-Aldrich	15,456-3