Introduction

1. Effect of energy source during culture on in vitro embryodevelopment, resistance tocryopreservation and sex ratio

2. Introduction • Despite many efforts to improve embryo culturesystems in different species, the oviduct remains irreplaceablefor embryo development. • Differences between in vivo and in vitroproduced embryos have been reported in metabolic andmorphologic profiles at the cellular level [1,2,3], aswell as in gene expression [4,5], resistance to cryopreservation[6] and post-transfer viability [7]

3. It is known that the environment has an impact on the developmental physiology of the embryos. Of the different steps of the in vitro production, embryo culture is likely to have the major influence, as embryos are held in media for up to eight days. • It is also known that culture conditions influence the sex ratio [9,10]. • Indeed, another limiting factor of IVEP consists in the alteration of the sex ratio towards males that is often recorded in the in vitro system [11–13].

4. Most of the embryo culture systems include glucose as the main energy source. • Alterations of metabolic profile have been reported in embryos cultured in the presence of glucose at blood serum concentrations [16]. • embryos can metabolize glucose from the zygote to the blastocyst stage [2,3,17–19]; however, limited amounts of glucose are oxidized by the tricarboxylic-acid cycle whereas the major fate of the hexose is lactate production [18,20]. The high lactate production associated to glycolytic activity [2] has been reported to reduce embryo development and viability [21].

5. Interestingly, the presence of glucose during IVC inhibits the development into blastocyst of female embryos more than that of the male counterparts [10], indicating a selective embryotoxicity towards females. • In standard tissue culture medium (TCM) glucose is present at the concentration found in human serum, i.e., 5.6 mM [24]. whereas in defined culture media, such as standard SOF [25], the concentration of glucose is 1.5 mM.

6. In a preliminary work, we demonstrated that bovine blastocysts can be produced in a medium containing minimum concentration of glucose and enriched with glyceraldehyde 3-phosphate (G3P), an intermediate metabolic compound of glycolysis, that enters directly the pay-off phase of the glycolytic pathway [32].

7. Therefore, the aim of this work was to evaluate whether minimizing the glucose concentration in the medium or replacing the hexose with other energy substrates and/or embryotrophic compounds, in a static culture system, would affect the in vitro embryo development, the resistance to cryopreservation and the sex ratio.

8. compared 4 different culture systems: • 1) 1.5 Mm glucose, as in standard SOF; • 2) 0.15 mM glucose; • 3) 0.125 mM G3P, in the presence of 0.15 mM glucose • 4) 0.34 mM citrate, in combination with 2.77 mMmyo-inositol.

9. Material and methods • Reagents and media • The aspiration mediumwas TCM 199 supplemented with 25 mMHepes, 2 mMsodium bicarbonate, 2 mM sodium pyruvate, 1 mML-glutamine, 10 L/mLamphotericin B (H199) supplementedwith 2% bovine serum (BS) and 95.6 SI/mLheparin. • The in vitro maturation (IVM) medium wasTCM 199 supplemented with 15% bovine serum (BS),0.5 g/mL FSH, 5 g/mL LH, 0.8 mML-glutamineand 50 g/mLgentamycin.

10. The IVF medium was Tyrode’s modified medium[33] without glucose and bovine serum albumin (BSA),supplemented with 5.3 SI/mL heparin, 30 M penicillamine,15 M hypotaurine, 1 M epinephrine and 1%of BS. • The IVC medium consisted of Synthetic OviductFluid (SOF) medium [25], with 30 L/mLessentialamino acids, 10 L/mL non essential amino acids and5 % BS, that was supplemented with different energysubstrates in each experimental group, as described insubsection 2.5.

11. The base medium for the vitrification and warmingsolutions was H199 with 20% fetal calf serum (FCS). • The equilibration medium (VS1) consisted of 7.5%DMSO and 7.5% ethylene glycol (EG) in base mediumand the vitrification solution (VS2) consisted of 16.5%DMSO and 16.5% EG with 0.5M sucrose in the basemedium. • The warming solutions consisted in 0.25 Mand 0.15 M sucrose in the base medium.

12. In vitro embryo production • Cumulus-oocyte complexes (COCs) wereaspirated • Groups of25 COCs were matured in 400 L of IVM medium,covered with mineral oil, in four well plates • COCs were washed and transferred,25 per well, into 300 L of IVF medium covered withmineral oil. • The sperm were added in the fertilization wells at the concentration of 1 106 sperm/mL.

13. randomly distributed (30–50 per well, into 400 L of IVC medium) into 4 culture groups differing from each other in the energy substrate. • After Incubation: On Day 7 (Day 0 IVF day) cleavage and blastocyst rates were recorded

14. Vitrification and warming • The blastocysts produced were vitrified by the Cryotop method • For warming, after removal of the cap, the Cryotopstrip was immersed directly into 1 mL of the 0.25 M sucrose solution for 1 min and the embryo was transferred into 200 L-droplet of 0.15 M sucrose solution for 5 min and then they were washed. • Survival rates were evaluated.

15. Embryo sexing • PCR products were analyzed using a 2% agarosegel with 0.1 g Ml-1 of ethidium bromide in the gel. The bands migrated by electrophoresis and the products were observed with UV transillumination.

16. Experimental design • Following IVF presumptive zygotes (n 2625) were randomly assigned to 4 experimental groups in which the IVC medium was supplemented with: • 1.5 mM glucose (group A; n 851); • 0.15 mM glucose (group B; n 554); 0.125 mM G3P in the presence of • 0.15 mM glucose (group C; n 608), and • 0.34 mMtri-sodium citrate 2.77 mM myo-inositol (group D; n 612).

17. The efficacy of the different culture systems to support embryo development was evaluated by comparing the percentages of Grade 1 and 2 blastocysts (Bl) in relation to total COCs, as well as the percentages of advanced embryos, i.e., expanded and hatched blastocysts (XBland HBL, respectively) out of the total blastocystsproduced, by the end of culture. • the survival and developmental rates of blastocysts were evaluated after 24 h culture.

18. Statistical analysis • Differences among groups in th percentages of blastocysts, of advanced embryos out of the total produced, as well as in the survival rates after vitrification warming, were analyzed by Chi square test.

19. Results

20. Differences in cleavage rates were observed among groups, with the highest valu recorded in group B (P 0.05). • With regard to post-fertilization embryo development, blastocyst rate was lower (P 0.01) in group A than in the other three groups, that gave similar values. • However, the proportion of oocytes reaching the most advanced stages of development (XBl and HBL) by the end of culture decreased in group D (11.4%) compared to both group A (15.8%; P 0.05) and groups B and C (18.8 and 20.3, respectively; P 0.01). • The percentage of advanced embryos out of the total blastocysts was also lower (P 0.05) in group B compared to groups A and C

22. the blastocysts produced in group D showed the worst resistance to cryopreservation, as indicated by the reduced (P 0.01) percentage of embryo survival, evaluated on morphological basis, after 24 h culture post-warming. • in group D the development rate, i.e., the percentage of blastocyst reaching a more advanced stage of development after in vitro culture post-warming was lower (P 0.05) than in group B, with groups A and C showing intermediate values.

23. The energy substrate affected the sex ratio of the embryos produced. • a higher (P 0.05) percentage of female embryos was obtained in group D (59/96 61.5%) compared to group A (68/149 45.6%), whereas intermediate values were found in groups B (62/116 53.4%) and C (64/128 50%).

24. Discussion • The first finding of the present study was that cleavage rate was significantly improved when glucose concentration was reduced up to 0.15 mM, i.e., 1/10 of the standard concentration in SOF (group B). Although sperm penetration has already occurred by the time the presumptive zygotes are transferred into IVC, it is likely that if they encounter an adverse or suboptimal environment the first cleavage division may be impaired.

25. In fact, both reducing glucose concentration in the presence (group C) or absence of G3P (group B) and replacing the hexose with citrate and myo-inositol (group D) significantly improved blastocyst production compared to standard SOF containing 1.5 mM glucose (group A). Also the percentage of cleaved oocytes developing into blastocysts was reduced in group A compared to the other groups.

26. Culture conditions have a major influence on survival an development of in vitro-produced embryos following cryopreservation. • the slower kinetics of embryo development, together with the poorer cryotolerance indicate a reduced viability of the embryos cultured in group D.

27. It is known that in the in vitro system the sex ratio is altered towards males and that male embryos develop in vitro faster than female embryos . • the sex ratio was altered towards males only in the group with higher concentration of glucose (group A), whereas an increased percentage of female embryos was achieved in group D, i.e., the only system that did not include glucose, apart for the very small amount present in the BS.

28. When glucose concentration was reduced, both in the presence(group C) and absence (group B) of G3P, the sex ratio was not skewed, with approximately 50% o embryosofeach gender. • supplementation of culture media with blood plasma-like glucose concentrations causes a preferential loss of female embryos during culture to the blastocyststage. • It results that glucose exerts a selective embryotoxicity towards female embryos at a concentration lower than 2 mM. • The higher vulnerability of female embryos to suboptimal culture conditions may be due to the unbalanced expression of X-linked genes before X inactivation occurs

29. The higher vulnerability of female embryos to suboptimal culture conditions may be due to the unbalanced expression of X-linked genes before X inactivation occurs. • Both the lower resistance to cryopreservation and the slower development of the embryos produced in group D may be related to the higher incidence of female embryos. In fact, previous reports have demonstrated that female embryos show a slower developmental rate and are more sensitive to cryopreservation.

30. These results suggest that manipulating the metabolic profile of the embryos during culture may have an impact on sex ratio.