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

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.