Development to the blastocyst stage of parthenogenetically activated in vitro matured porcine oocytes after solid surface vitrification (SSV)

The effects of solid surface vitrification (SSV) on viability and parthenogenetic development of in vitro matured (IVM) porcine oocytes was investigated in the present study. Cumulus-free IVM porcine oocytes were subjected either to SSV or SSV combined with a cytochalasin B (CB) pre-treatment (SSV+CB) or all steps of SSV but without cooling (toxicity control=TC; toxicity control with CB pre-treatment=TC+CB). Oocyte viability was evaluated by plasma membrane integrity and esterase activity measured by a combined staining with fluorescein diacetate, propidium iodide and Hoechst 33342. Surviving oocytes were parthenogenetically activated then cultured in vitro (IVC) for 6 days. The proportion of live oocytes after vitrification was significantly lower than that of the TC, TC+CB and the control groups, regardless of the CB pre-treatment. Treatment of oocytes with cryoprotectants did not decrease the rates of surviving oocytes. After activation of oocytes, the proportion of cleaved embryos was significantly higher in the SSV+CB (P<0.05) than that of the SSV group. Nevertheless, significantly more oocytes cleaved (P<0.05) in the TC, TC+CB and the control groups. On Day 6, the rate of blastocysts in the SSV and SSV+CB groups did not differ significantly. The number of oocytes developing to blastocyst and the mean number of blastomeres per embryo were significantly higher (P<0.05) in the TC, TC+CB and the control compared with that of the SSV and SSV+CB groups. To our knowledge, this is the first report on parthenogenetic development to blastocysts of porcine oocytes vitrified at the metaphase-II stage. Results indicate that the high concentrations of cryoprotectants were not harmful for in vitro development, and that CB pre-treatment may increase survival and development of SSV vitrified porcine oocytes.     

Farrowing rates and litter size following transfer of vitrified porcine embryos into a commercial swine herd

The objective was to determine farrowing rates and litter sizes that could be achieved in a typical farm-to-farm porcine embryo transfer program using vitrified blastocysts that were zona pellucida intact when cryopreserved. The embryos were transferred surgically on-farm into recipient sows that were managed throughout gestation and farrowing under the same conditions as other sows in the herd. Twenty recipient sows (mean parity 2.1) received a total of 568 embryos; seven received 203 embryos derived from donor sows, five received 139 embryos from gilts and eight received a mixture of 161 embryos from sows and 65 from gilts. Sixteen sows (80%) were confirmed pregnant at approximately 35 days gestation, 15 farrowed at full term (farrowing rate 75%). One sow died during gestation (with a total of 18 fetuses in utero). A total of 123 piglets were born (mean, 8.2), of which 115 were born alive (mean, 7.7). Of the 568 embryos transferred to all 20 sows, 21.6% resulted in piglets born and 29.0% survived to produce piglets in sows that farrowed. There were no significant differences in embryo survival among sow, gilt or mixed sow and gilt embryos. The ratio of males to females was 71/52 and the mean birth weight was 1.6 kg (range 0.6-2.6 kg). In conclusion, vitrified zona pellucida intact embryos can be used to transfer genetic material from farm-to-farm with acceptable reproductive performance.     

An epigenetic modifier results in improved in vitro blastocyst production after somatic cell nuclear transfer

The present study was designed to examine the effect of trichostatin A (TSA), an inhibitor of histone deacetylase, on development of porcine cloned embryos. Our results showed that treatment of cloned embryos derived from sow oocytes with 50 nM TSA for up to 24 h after the onset of activation could significantly improve blastocyst yield compared to the control (46.4+/-4.6% vs 17.7+/-4.9% for treated and untreated embryos, respectively; p<0.05), whereas similar cleavage rate and total cell number per blastocyst were observed. In order to assess if the improvement is cell line specific, three cell lines were tested, and for all cell lines an enhancement in blastocyst development compared to their corresponding control was observed. Our data demonstrate that TSA treatment after somatic cell nuclear transfer in the pig can significantly improve the in vitro blastocyst production.     

Proteomic analysis of reproduction proteins involved in litter size from porcine placenta

A gel-free and label-free quantitative proteomic approach based on a spectral counting strategy was performed to discover prolificacy-related proteins. Soluble proteins of porcine placenta from small litter size group (SLSG) and large litter size group (LLSG) were extracted and subsequently applied to in-solution tryptic digestion followed by liquid chromatography–tandem mass spectrometry analysis. Six and thirteen proteins were highly expressed in SLSG and LLSG, respectively. Of the dominantly expressed proteins, we chose prolificacy-related proteins such as puromycin-sensitive aminopeptidase (PSA) and retinol-binding protein 4 (RBP4). Western blot analysis confirmed that the processed form (70 kDa) of PSA was more expressed and RBP4 (23 kDa) was dominantly expressed in LLSG. These results indicate that PSA and RBP4 are representative proteins involved in porcine fertility traits, and this finding may help to increase litter size of pigs.     

Correlated responses on litter size traits and survival traits after two-stage selection for ovulation rate and litter size in rabbits

Farmer profit depends on the number of slaughter rabbits. The improvement of litter size (LS) at birth by two-stage selection for ovulation rate (OR) and LS could modify survival rate from birth to slaughter. This study was aiming to estimate direct and correlated response on LS traits and peri- and postnatal survival traits in the OR_LS rabbit line selected first only for OR (first period) and then for OR and LS using independent culling levels (second period). The studied traits were OR, LS measured as number of total born, number of kits born alive (NBA) and dead (NBD), and number of kits at weaning (NW) and young rabbits at slaughter (NS). Prenatal survival (LS/OR) and survival at birth (NBA/LS), at weaning (NW/NBA) and at slaughter (NS/NW) were also studied. Data were analysed using Bayesian inference methods. Heritability for LS traits were low, 0.07 for NBA, NW and NS. Survival traits had low values of heritability 0.07, 0.03 and 0.03 for NBA/LS, NW/NBA and NS/NW, respectively. After six generations of selection by OR (first period), a small increase in NBD and a slight decrease in NBA/LS were found. However, no correlated responses on NW/NBA and NS/NW were observed. After 11 generations of two-stage selection for OR and LS (second period), correlated responses on NBA, NW and NS were 0.12, 0.12 and 0.11 kits per generation, respectively, whereas no substantial modifications on NBA/LS, NW/NBA and NS/NW were found. In conclusion, two-stage selection improves the number of young rabbits at slaughter without modifying survival from birth to slaughter.     

Genome-enabled methods for predicting litter size in pigs: a comparison

Predictive ability of models for litter size in swine on the basis of different sources of genetic information was investigated. Data represented average litter size on 2598, 1604 and 1897 60K genotyped sows from two purebred and one crossbred line, respectively. The average correlation (r) between observed and predicted phenotypes in a 10-fold cross-validation was used to assess predictive ability. Models were: pedigree-based mixed-effects model (PED), Bayesian ridge regression (BRR), Bayesian LASSO (BL), genomic BLUP (GBLUP), reproducing kernel Hilbert spaces regression (RKHS), Bayesian regularized neural networks (BRNN) and radial basis function neural networks (RBFNN). BRR and BL used the marker matrix or its principal component scores matrix (UD) as covariates; RKHS employed a Gaussian kernel with additive codes for markers whereas neural networks employed the additive genomic relationship matrix (G) or UD as inputs. The non-parametric models (RKHS, BRNN, RNFNN) gave similar predictions to the parametric counterparts (average r ranged from 0.15 to 0.23); most of the genome-based models outperformed PED (r = 0.16). Predictive abilities of linear models and RKHS were similar over lines, but BRNN varied markedly, giving the best prediction (r = 0.31) when G was used in crossbreds, but the worst (r = 0.02) when the G matrix was used in one of the purebred lines. The r values for RBFNN ranged from 0.16 to 0.23. Predictive ability was better in crossbreds (0.26) than in purebreds (0.15 to 0.22). This may be related to family structure in the purebred lines.     

Effects of caffeine treatment on aged porcine oocytes: parthenogenetic activation ability, chromosome condensation and development to the blastocyst stage after somatic cell nuclear transfer

The possibility of using aged porcine oocytes treated with caffeine, which inhibits the decrease in M-phase promoting factor activity, for pig cloning was evaluated. Cumulus-oocyte complexes (COCs) were cultured initially for 36 h and subsequently with or without 5 mM caffeine for 24 h (in total for 60 h: 60CA+ or 60CA- group, respectively). As a control group, COCs were cultured for 48 h without caffeine (48CA-). The pronuclear formation rates at 10 h after electrical stimulation in the 60CA+ and 60CA- groups decreased significantly (p < 0.05) compared with the 48CA- group. However, the fragmentation rate was significantly higher (p < 0.05) in the 60CA- group than in the 60CA+ and 48CA- groups. When the stimulated oocytes were cultured for 6 days, the 60CA+ group showed significantly lower blastocyst formation and higher fragmentation or degeneration rates (p < 0.05) than the 48CA- group. However, the number of total cells in blastocysts was not affected by maturation period or caffeine treatment. When somatic cell nuclei were injected into the non-enucleated oocytes and exposed to cytoplasm for a certain duration (1-11 h) before the completion of maturation (48 or 60 h), the rate of nuclear membrane breakdown after exposure to cytoplasm for 1-2 h in the 60CA- oocytes was significantly lower (p < 0.05) than in the other experimental groups. The rate of scattered chromosome formation in the same 60CA- group tended to be lower (p = 0.08) than in the other groups. After the enucleation and transfer of nuclei, blastocyst formation rates in the 60CA+ and 60CA- groups were significantly lower (p < 0.05) than in the 48CA- group. Blastocyst quality did not differ among all the groups. These results suggest that chromosome decondensation of the transplanted somatic nucleus is affected by both the duration of exposure to cytoplasm and the age of the recipient porcine oocytes, and that caffeine treatment promotes nuclear remodelling but does not prevent the decrease in the developmental ability of cloned embryos caused by oocyte aging.     

Changes in litter size in Kerry Blue Terrier dogs with abnormal dentition

The pleiotropic effects of mutations resulting in abnormal dentition were analyzed in Kerry Blue Terrier. A decrease in litter size was demonstrated for dogs with dentition anomalies. The mean litter size was 5.72 puppies when both parents had normal dentition and 3.64 puppies when the parents had hypodontia. Analysis showed that the decrease in litter size cannot be fully explained by the effect of inbreeding and is most probably associated with the pleiotropic effect of the genes controlling teeth development on the embryonic viability.     

Effect of estrous cow serum during bovine embryo culture on blastocyst development and cryotolerance after slow freezing or vitrification

The present study investigated the effect of estrous cow serum (ECS) during culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. Embryos were derived from in vitro maturation (IVM) and in vitro fertilization (IVF) of abbatoir-derived oocytes. At Day 3, embryos were cultured in three different media: Charles Ronsenkrans medium + amino acids (CR1aa; without bovine serum albumin (BSA)) + 5% estrous cow serum (CR1-ECS), CR1aa + 3 mg/mL BSA (CR1-BSA) or CR1aa + 5% ECS + 3 mg/mL BSA (CR1-ECS-BSA). At 7.5 d post-insemination (PI), blastocyst yield and quality were evaluated; blastocysts and expanded blastocysts from each media were cryopreserved by Open Pulled Straw (OPS) vitrification method or slow freezing (1.5 M ethylene glycol, EM). Total blastocyst yield did not differ among CR1-ECS, CR1-BSA and CR1-ECS-BSA (30.9, 33.1 and 32.9%, respectively, P < 0.05). Embryo survival (hatching rate) was higher in vitrified versus slow-frozen embryos (43% versus 12%, respectively, P < 0.01), and in embryos cultured in CR1-BSA (40.3%) compared with those cultured in serum-containing media (CR1-ECS, 21.5% and CR1-ECS-BSA, 19.8%; P < 0.01). In conclusion: (a) it was possible to produce in vitro bovine embryos in serum-free culture medium without affecting blastocyst yield and quality; (b) serum-free medium produced the best quality embryos (in terms of post-cryopreservation survival); and (c) vitrification yielded the highest post-cryopreservation survival rates, regardless of the presence of serum in the culture medium.     

Selection for fertility in mice using different methods of litter size manipulation

Summary A short-term selection experiment for increasing the first-day litter size (LS1) and 28-day litter weight (LW28) was conducted with three populations of mice over 8 generations. Different methods of litter size manipulation were used for the populations — in S the litter size was standardized to 8 (4 , 4 ) on the first day, in LA it was adjusted to the average size of all litters born on the same day and NL had the natural litter size. To eliminate temporary environmental effects, a control population was kept in each case. The selection results per generation were, for LS 1 b=0.30 (S, NL) and 0.20 (LA), and for LW28 b=5.62 g (S), 5.26 g (NL), and 4.32 g (LA). The heritability obtained was between 0.18 and 0.13 for LS 1 and from 0.42 to 0.12 for LW28. The populations differed in the correlated responses for body weight parameters (litter weight gain). The implantation rate increased in populations S and NL (b=0.19, 0.37), but not in population LA. Postnatal mortality went down (b=-0.07) and the dam's milk production rose (b=1.11 g) only in population LA. The estimated partial regression coefficient linking body weight at mating (BWM) for the dam and the daughter's litter size showed an effect on the litter size.     

Uncoupled embryonic and extra-embryonic tissues compromise blastocyst development after somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each); one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular "uncoupling". Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity) and subsequent pregnancy loss. Finally, because it alters re-differentiation processes in vivo, SCNT reprogramming highlights temporally and spatially restricted interactions among cells and tissues in a unique way.     

Changes in rat milk quantity and quality due to variations in litter size and high ambient temperature.

The effects of variations in suckling stimulus (changes in letter size) and exposure to high ambient temperatures on the quality and quantity of rat's milk and mammary tissue structure were determined. By increasing the number of suckling pups per dam, it was found that the amount of milk produced by the animals was proportionally increased, as each pup was able to drink the same amount of milk per day. With more than 6 pups per litter, the quality of the milk was not significanly changed. Smaller letters led to changes in the amount of milk suckled per milking and to considerable changes in the content of the milk. Exposure to high ambient temperatures, although for only 8 hr per day, drastically affected the amount of milk produced by the lactating rat as well as the quality of the milk produced. Microscopic examination of mammary tissue revealed changes in the alveolar area and height of the alveolar cells as the suckling stimulus varied. After heat exposure the gland changed to one of diminished synthesizing capability. It was concluded that 10-pup litters are the optimal litter size for research concerning rat milk, and the exposure to high ambient temperatures affects the milk yield by directly affecting the alveolae.     

Changes in litter size in Kerry blue terrier dogs with abnormal dentition

The pleiotropic effects of mutations resulting in abnormal dentition were analyzed in Kerry Blue Terrier. A decrease in litter size was demonstrated for dogs with dentition anomalies. The mean litter size was 5.72 puppies when both parents had normal dentition and 3.64 puppies when the parents had hypodontia. Analysis showed that the decrease in litter size cannot be fully explained by the effect of inbreeding and is most probably associated with the pleiotropic effect of the genes controlling teeth development on the embryonic viability.     

Birth of piglets after transfer of embryos cryopreserved by cytoskeletal stabilization and vitrification

Pig embryos suffer severe sensitivity to hypothermic conditions, which limits their ability to withstand conventional cryopreservation. Research has focused on high lipid content of pig embryos and its role in hypothermic sensitivity, while little research has been conducted on structural damage. Documenting cytoskeletal disruption provides information on embryonic sensitivity and cellular response to cryopreservation. The objectives of this study were to document microfilament (MF) alterations during swine embryo vitrification, to utilize an MF inhibitor during cryopreservation to stabilize MF, and to determine the developmental competence of cytoskeletal-stabilized and vitrified pig embryos. Vitrified morulae/early blastocysts displayed MF disruptions and lacked developmental competence after cryopreservation; hatched blastocysts displayed variable MF disruption and developmental competence. Cytochalasin-b did not improve morula/early blastocyst viability after vitrification; however, it significantly (P < 0.05) improved survival and development of expanded and hatched blastocysts. After embryo transfer, we achieved pregnancy rates of almost 60%, and litter sizes improved from 5 to 7.25 piglets per litter. This study shows that the pig embryo cytoskeleton can be affected by vitrification and that MF depolymerization prior to vitrification improves blastocyst developmental competence after cryopreservation. After transfer, vitrified embryos can produce live, healthy piglets that grow normally and when mature are of excellent fecundity.     

Resveratrol improved the developmental potential of oocytes after vitrification by modifying the epigenetics

Resveratrol (Res) has been reported to be able to improve oocyte vitrification because of its antioxidative properties. The objective of this study was to further assess the positive effect of Res addition on the developmental potential of vitrified mouse oocytes from the perspective of epigenetic alterations. First, 2 μM Res was chosen as the optimal concentration on the basis of its effects on survival and its antioxidative properties. We found that Res addition significantly promoted fertilization (63.8% vs. 42.9%) and blastocyst formation (68.3% vs. 50.2%) after oocyte vitrification. The quality of the derived blastocysts was also higher after Res treatment. Regarding epigenetic aspects, the expression of the important deacetylase SIRT1 was found to decrease significantly upon vitrification, but it was rescued by Res. The abnormal levels of H3K9 acetylation and DNA methylation in vitrified oocytes were restored by Res addition. Moreover, the expression of several imprinted genes was affected by oocyte vitrification. Among them, abnormal Gtl2 and Peg3 expression levels were restored by Res addition. Therefore, the methylation of their imprinted control regions (ICRs) was examined. Surprisingly, the abnormal patterns of Gtl2 and Peg3 methylation in blastocysts developed from vitrified oocytes were both restored by Res addition. Finally, the full‐term embryonic development showed that the birth rate was improved significantly by Res addition (56.2% vs. 38.1%). Collectively, Res was beneficial for the pre‐ and postimplantation embryonic development. Except for the antioxidative activity, Res also played a role in the correction of some abnormal epigenetic modifications caused by oocyte vitrification.     

A new selection criterion to assess good quality ovine blastocysts after vitrification and to predict their transfer into recipients

The feasibility to accurately select viable embryos would be valuable for improving pregnancy rates and avoiding futile transfer attempts. The aim of our study was to assess if in vitro-produced embryo quality could be determined by the timing of blastocoelic cavity re-expansion after vitrification, warming, and in vitro culture using sheep as a model. Blastocysts were produced in vitro, vitrified/warmed, and cultured in TCM-199 plus 10% FCS for 72 hr. Embryos were divided into two groups: re-expanded within 8 hr (A) and from 8 to 16 hr (B) of IVC after warming. Fast re-expanded blastocysts showed higher in vitro hatching rates and total cell number calculated on the hatched blastocysts compared with slow re-expanded ones (P < 0.01). Peroxide status evaluation (P < 0.01) and TUNEL test (P < 0.05) revealed a higher number of positive cells in group B compared with group A. The quantitative analysis of protein synthesis revealed a higher synthesis in fast compared with slow re-expanded embryos (P < 0.05). Quantitative RT-PCR showed that 90-kDa Heat Shock Protein beta was more expressed in group A than in group B (P < 0.05), while the quantity of P34(cdc2), Cyclin b, Aquaporin 3, Na/K ATPase, and Actin did not differ between the two groups. Pregnancy rates after transfer to synchronized recipients were higher in fast compared to slow re-expanded blastocysts (P < 0.05). Our results evidenced that timing of blastocoelic cavity re-expansion after vitrification/warming and in vitro culture can be considered as a reliable index of in vitro produced embryo quality and developmental potential.