Antifreeze protein type I in the vitrification solution improves the cryopreservation of immature cat oocytes.

Oocyte cryopreservation is not yet considered a reliable technique since it can reduce the quality and survival of oocytes in several species. This study determined the effect of different concentrations of antifreeze protein I (AFP I) on the vitrification solution of immature cat oocytes. For this, oocytes were randomly distributed in three groups and vitrified with 0 µg/mL (G0, 0 µM); 0.5 µg/mL (G0.5, 0.15 µM), or 1 µg/mL (G1, 0.3 µM) of AFP I. After thawing, oocytes were evaluated for morphological quality, and compared to a fresh group (FG) regarding actin integrity, mitochondrial activity and mass, reactive oxygen species (ROS) and glutathione (GSH) levels, nuclear maturation, expression of GDF9, BMP15, ZAR-1, PRDX1, SIRT1, and SIRT3 genes (normalized by ACTB and YWHAZ genes), and ultrastructure. G0.5 and G1 presented a higher proportion of COCs graded as I and while G0 had a significantly lower quality. G1 had a higher percentage of intact actin in COCs than G0 and G0.5 (P < 0.05). There was no difference (P > 0.05) in the mitochondrial activity between FG and G1 and they were both higher (P < 0.05) than G0 and G0.5. G1 had a significantly lower (P < 0.05) mitochondrial mass than FG and G0, and there was no difference among FG, G0, and G0.5. G1 had higher ROS than all groups (P < 0.05), and there was no difference in GSH levels among the vitrified groups (P > 0.05). For nuclear maturation, there was no difference between G1 and G0.5 (P > 0.05), but these were both higher (P < 0.05) than G0 and lower (P < 0.05) compared to FG. Regarding gene expression, in G0 and G0.5, most genes were downregulated compared to FG, except for SIRT1 and SIRT3 in G0 and SIRT3 in G0.5. In addition, G1 kept the expression more similar to FG. Regardless of concentration, AFP I supplementation in vitrification solution of immature cat oocytes improved maturation rates, morphological quality, and actin integrity and did not impact GSH levels. In the highest concentration tested (1 µg/mL), AFP maintained the mitochondrial activity, reduced mitochondrial mass, increased ROS levels, and had the gene expression more similar to FG. Altogether these data show that AFP supplementation during vitrification seems to mitigate some of the negative impact of cryopreservation improving the integrity and cryosurvival of cat oocytes.

Role of phosphoinositide 3-kinase/ protein kinase B/ phosphatase and tensin homologue (PI3K/AKT/PTEN) pathway inhibitors during in vitro maturation of mammalian oocytes on in vitro embryo production: A systematic review.

Modulation of phosphoinositide 3-kinase/protein kinase B/phosphatase and tensin homologue (PI3K/AKT/PTEN) pathway in mammals yields mixed results. A deep understanding of its regulation can be a powerful tool for better in vitro blastocyst production. This systematic review aims to map the evidence of PI3K/AKT/PTEN pathway modulation during in vitro maturation (IVM), to assess its effects on meiosis resumption and nuclear maturation progression of mammalian oocytes, and their impacts on embryo development and quality. A total of 1058 articles were screened in three databases, and 22 articles were included. Fifty-two IVM assessments were identified, among which 11 evaluated blastocyst yield. Three PI3K inhibitors (3-methyladenine, Wortmannin, and LY294002) and one AKT inhibitor (SH6) were investigated. The impact of this pathway modulation on meiosis resumption in swines and murines was not well established, depending on the inhibitor used, concentration, and media supplementation, while in bovines, resumption seems to be independent of PI3K/AKT/PTEN pathway. However, progression to metaphase II (MII) is highly controlled by this pathway on both bovines and swines. Studies that focused on the inhibition reversibility showed that the removal of the modulator produced MII rates similar to the control group. Experiments that aimed to temporarily block meiosis resumption or reduce PI3K activity resulted in blastocyst production equal to or even higher than control groups. Altogether, these data indicate the paramount potential of this pathway as a possible strategy to improve overall in vitro embryo production efficiency, by synchronizing both nuclear and cytoplasmic maturation.

Embryo development is impaired in goats that are treated for hydrometra and subsequently subjected to superovulation.

BACKGROUND: Reproductive efficiency after hydrometra (HD) treatment is usually unsatisfactory. METHODS: To identify mechanisms involved in low reproductive efficiency of HD-treated goats, pluriparous dairy goats treated for HD (n=10, HD) or with no reproductive disorders (n=11, control: CONT) were induced to oestrus and superovulated. Goats were mated with fertile bucks and seven days after oestrus, non-surgical embryo recovery was performed. Embryos were evaluated and gene expression was performed. RESULTS: There were no differences (P>0.05) in sexual behaviour parameters, superovulation response, mean number of retrieved structures and viable embryos between groups; although embryo recovery rate was higher (P=0.01) in CONT group. Structures in delayed stage (8-16 cells) were more frequent (P<0.05) in HD (29 vs 1 per cent) goats, as well as the percentage of advanced embryos was greater (P<0.05) for CONT (59.3 vs 33.3 per cent) goats. However, the expression of genes related to apoptosis (BAX and Bcl-2), trophectoderm differentiation (CDX2) and pluripotency maintenance (NANOG) was not affected (P>0.05) in embryos that reached the morulae and blastocyst stages. CONCLUSION: Although the HD embryos that developed to morula and blastocyst stages showed no change in the expression of genes related to their quality and implantation capacity, overall, embryo development was impaired in HD-treated goats.

Comparison of the intravenous and intravaginal route of oxytocin administration for cervical dilation protocol and non-surgical embryo recovery in oestrous-induced Santa Inês ewes.

This study compared the effects of intravaginal and intravenous routes of oxytocin (OT) administration in 46 oestrous-induced Santa Inês ewes (6-day treatment with progestin-releasing intravaginal sponges and a single injection of 200 IU of eCG at the time of sponge removal) that underwent transcervical embryo recovery 6-7 days after oestrous onset and mating. All ewes received 37.5 µg of d-cloprostenol via latero-vulvar route, and 1 mg of oestradiol benzoate i.m. 16 hr before and 50 IU of OT 20 min before non-surgical embryo recovery (NSER), with OT being administered intravenously (n = 21) or intravaginally (n = 21). An overall oestrous response was 95.6% (44/46), and adequate cervical retraction could be accomplished in 78.6% (33/42) of ewes. The percentage of successful NSER procedures was 57% (24/42) or 72.7% (24/33) of animals with sufficient cervical retraction. The duration of NSER procedure averaged 28 min (range: 17-40 min) and ~96% of flushing fluid could be recovered (range: 85%-100%). Out of 18 ewes that could not undergo NSER, 12 (66.6%) presented various anatomical barriers, whilst the other 33.4% did not present these barriers and still could not be traversed. Excluding the ewes with those anatomical features, the overall success rate of NSER was 80% (24/30). The route of OT administration had no effect on NSER efficiency or the ease with which transcervical embryo flushing was performed. Both routes of OT administration can be used for cervical dilation protocol. Discarding ewes with anatomical features precluding cervical penetration is highly recommended to increase the efficacy of NSER in sheep.