Equine chorionic gonadotropin and gonadotropin-releasing hormone enhance fertility in a norgestomet-based, timed artificial insemination protocol in suckled Nelore (Bos indicus) cows

Two experiments were conducted to investigate the effects of equine chorionic gonadotropin (eCG) at progestin removal and gonadotropin-releasing hormone (GnRH) at timed artificial insemination (TAI) on ovarian follicular dynamics (Experiment 1) and pregnancy rates (Experiment 2) in suckled Nelore (Bos indicus) cows. Both experiments were 2 × 2 factorials (eCG or No eCG, and GnRH or No GnRH), with identical treatments. In Experiment 1, 50 anestrous cows, 134.5 ± 2.3 d postpartum, received a 3 mg norgestomet ear implant sc, plus 3 mg norgestomet and 5 mg estradiol valerate im on Day 0. The implant was removed on Day 9, with TAI 54 h later. Cows received 400 IU eCG or no further treatment on Day 9 and GnRH (100 μg gonadorelin) or no further treatment at TAI. Treatment with eCG increased the growth rate of the largest follicle from Days 9 to 11 (means ± SEM, 1.53 ± 0.1 vs. 0.48 ± 0.1 mm/d; P < 0.0001), its diameter on Day 11 (11.4 ± 0.6 vs. 9.3 ± 0.7 mm; P = 0.03), as well as ovulation rate (80.8% vs. 50.0%, P = 0.02), whereas GnRH improved the synchrony of ovulation (72.0 ± 1.1 vs. 71.1 ± 2.0 h). In Experiment 2 (n = 599 cows, 40 to 120 d postpartum), pregnancy rates differed (P = 0.004) among groups (27.6%, 40.1%, 47.7%, and 55.7% for Control, GnRH, eCG, and eCG + GnRH groups). Both eCG and GnRH improved pregnancy rates (51.7% vs. 33.8%, P = 0.002; and 48.0% vs 37.6%, P = 0.02, respectively), although their effects were not additive (no significant interaction). In conclusion, eCG at norgestomet implant removal increased the growth rate of the largest follicle (LF) from implant removal to TAI, the diameter of the LF at TAI, and rates of ovulation and pregnancy rates. Furthermore, GnRH at TAI improved the synchrony of ovulations and pregnancy rates in postpartum Nelore cows treated with a norgestomet-based TAI protocol.     

Pregnancy rates and corpus luteum-related factors affecting pregnancy establishment in bovine recipients synchronized for fixed-time embryo transfer

The objective was to investigate the influence of corpora lutea physical and functional characteristics on pregnancy rates in bovine recipients synchronized for fixed-time embryo transfer (FTET). Crossbred (Bos taurus taurus × Bos taurus indicus) nonlactating cows and heifers (n = 259) were treated with the following protocol: 2 mg estradiol benzoate (EB) plus an intravaginal progesterone device (CIDR 1.9 g progesterone; Day 0); 400 IU equine chorionic gonadotropin (eCG; Day 5); prostaglandin F_2α (PGF_2α) and CIDR withdrawal (Day 8); and 1 mg EB (Day 9). Ovarian ultrasonography and blood sample collections were performed on Day 17. Of the 259 cattle initially treated, 197 (76.1%) were suitable recipients; they received a single, fresh, quality grade 1 or 2 in vivo-derived (n = 90) or in vitro-produced (n = 87) embryo on Day 17. Pregnancy rates (23 d after embryo transfer) were higher for in vivo-derived embryos than for in vitro-produced embryos (58.8% vs. 31.0%, respectively; P < 0.001). Mean (±SD) plasma progesterone (P₄) concentration was higher in cattle that became pregnant than that in nonpregnant cattle (5.2 ± 5.0 vs. 3.8 ± 2.4 ng/mL; P = 0.02). Mean pixel values (71.8 ± 1.3 vs. 71.2 ± 1.1) and pixel heterogeneity (14.8 ± 0.3 vs. 14.5 ± 0.5) were similar between pregnant and nonpregnant recipients (P > 0.10). No significant relationship was detected between pregnancy outcome and plasma P₄, corpus luteum area, or corpus luteum echotexture. Embryo type, however, affected the odds of pregnancy. In conclusion, corpus luteum-related traits were poor predictors of pregnancy in recipients. The type of embryo, however, was a major factor affecting pregnancy outcome.     

Embryo loss in cattle between Days 7 and 16 of pregnancy

Embryo loss between embryonic Days 7 and 16 (Day 0 = day of IVF) in nonlactating cattle, Bos taurus, was analyzed using transfer of 2449 (in groups of 3 to 30) in vitro-produced (IVP) blastocysts. In 152 transfers, pregnancy losses attributable solely to recipient failings amounted to between 6% (beef heifers) and 16% (parous dairy cows), of which 3% were caused by uterine infections. Neither season, year, nor the age of the embryos on retrieval affected pregnancy rates. The latter observation indicated that the reason that a recipient failed to retain embryos was already present at the time of transfer. Notably, the proportion of embryos recovered decreased (P = 0.03) as more embryos were transferred, particularly at later stages (Day 14, P < 0.01). The average length of embryos decreased by approximately 5% for every additional embryo transferred (P < 0.0001). These effects may be linked to embryonic migration. Embryo mortality inherent to the embryo during the second week of pregnancy was 24%. Additionally, 9% of Day 14 embryos were of inferior quality, as they did not contain an epiblast. Combining embryo and recipient causes but excluding infection effects, embryonic loss of IVP embryos during the second week of pregnancy amounted to 26% (heifers) or 34% (parous dairy cows). The length of embryos doubled every day between Days 9 and 16, with a 4.4-fold range in sizes representing two thirds of the variation in length. Embryos retrieved from heifers were twice the size of those incubated in parous cows (P < 0.0001), indicating faster embryonic development/trophoblast proliferation in heifers. Whereas season did not affect embryo recoveries, length was lower (50%) in winter (winter-autumn, P < 0.05; winter-spring, P < 0.001). Lastly, transuterine migration in cattle, when transferring multiple embryos, commenced at Day 14 (4%) and had occurred in all recipients by Day 16 (38% of embryos found contralaterally).     

In vitro development of embryos from superovulated gilts treated with the progesterone agonist, altrenogest (Regu-Mate) or the prostaglandin analogue, cloprostenol (Planate)

This study was conducted to compare the in vitro development of embryos from superovulated postpubertal gilts synchronized with progesterone agonist altrenogest (REG, Regu-Mate) and those from superovulated prepubertal gilts synchronized with prostaglandin analogue cloprostenol (PLA, Planate). Ten postpubertal gilts that had exhibited estrus at least once were fed 20 mg/d of REG from Day 0 (the first day of treatment, may have been any day of the estrous cycle) to Day 17. The gilts received intramuscularly (im) 1500 IU of equine chorionic gonadotropin (eCG) on the afternoon of Day 17, followed by 1000 IU of human chorionic gonadotropin (hCG) 84 h later. Eight prepubertal gilts received intramuscularly one dose of a combination of 400 IU of eCG and 200 IU of hCG (PG 600) on Day 0 (the first day of treatment), followed by 750 IU of hCG on Day 3. From Day 16 to Day 19, the prepubertal gilts received 350 mg/d of PLA, followed by 1500 IU of eCG on the afternoon of Day 19, then 1000 IU of hCG 84 h later. Gilts were checked for estrus with an intact boar. At estrus, all gilts were artificially inseminated and/or mated twice at 12-h intervals. Then 50 to 54 h after the hCG injection, a mid-ventral laparotomy was performed on each gilt. Corpora albicans (CA) and corpora hemorrhagica (CH) were counted, and oviducts were flushed in situ. The embryos recovered (1- to 2-cell) were cultured in modified Whitten's medium at 38.5 degrees C under an atmosphere of 5% CO2 in air for 144 h. The number of CA per gilt did not differ between the postpubertal and prepubertal gilts (11.9 vs 7.9, respectively; P > 0.05). However, the number of CH per gilt (27.5 vs 18.1, P = 0.05) and the number of embryos per gilt (26.2 vs 15.3, P < 0.05) were higher in postpubertal gilts than in prepubertal gilts. Furthermore, after 144 h of in vitro culture, the percentage of embryos cleaving to the >-16-cell (morula + blastocysts) or > or =32-cell (blastocysts) was greater (P < 0.05) in prepubertal gilts than in postpubertal gilts (85.2 vs 68.5, 55.7 vs 44.2, respectively). The total numbers of embryos examined were 122 and 260 in prepubertal and postpubertal gilts, respectively. These results show that postpubertal gilts treated with REG produced a higher number of embryos. However, better embryo development was noted with zygotes from prepubertal gilts primed with exogenous gonadotrophin, followed by synchronization with prostaglandin before induction of superovulation and insemination.     

Transforming growth factor-α in a defined medium during in vitro maturation of porcine oocytes improves their developmental competence and intracellular ultrastructure

We examined the effects of transforming growth factor-α (TGF-α) to develop a defined medium for in vitro maturation (IVM) of porcine (Sus scrofa domesticus) oocytes. Cumulus-oocyte complexes (COCs) were matured in porcine oocyte medium containing 3 mg/mL polyvinyl alcohol (POM) and TGF-α (0, 1, 10, or 100 ng/mL) in the presence or absence of the gonadotropins equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). In the absence of gonadotropins, adding 10 ng/mL TGF-α increased (P < 0.05) the percentage of oocytes that reached metaphase II (24.2%) compared with that of the control (no TGF-α addition; 5.6%). In the presence of gonadotropins, although maturation rate did not differ among TGF-α treatments (75.4% to 84.8%), the rate of blastocyst formation (28.1%) was higher (P < 0.05) in the TGF-α group (28.1%) than that in the control group (15.9%) after in vitro fertilization and embryo culture. An electron microscope study revealed that TGF-α–treated oocytes contained more homogenous lipid droplets than did control oocytes. Moreover, mitochondria surrounded by the endoplasmic reticulum were observed only in the TGF-α–treated oocytes. In blastocysts derived from the latter oocytes, mitochondria with numerous cristae were frequently observed compared with that in blastocysts from control oocytes. When the Day-5 blastocysts obtained from oocytes matured with TGF-α were surgically transferred into four recipients, a total of 29 piglets were farrowed. We concluded that the addition of TGF-α to the defined IVM medium of porcine oocytes improved the subsequent blastocyst formation and that the blastocysts produced by the defined in vitro production system have developmental competence to full term after embryo transfer.     

Influence of hormonal and nonhormonal estrus synchronization methods on follicular and oocyte quality in primiparous lactating does at early postpartum period

High-yield lactating does need effective estrus synchronization methods to improve their reproductive outcome by enhancing ovarian function. The aim of the current work was to analyze ovarian follicular and oocyte characteristics of hormonal and nonhormonal estrus synchronization regimes in primiparous lactating rabbit does (Oryctolagus cuniculus) in the early postpartum period (Day 11). Females were randomly treated with either (1) a hormonal standard treatment with 25 IU equine chorionic gonadotropin (eCG) 48 h before artificial insemination (eCG group) or (2) an alternative nonhormonal treatment consisting of doe-litter separation 24 h before artificial insemination (Bio group). No significant differences were found in serum estradiol and progesterone concentrations between experimental groups. During the histologic study, the Bio group presented a higher number of primordial (P < 0.05) and primary follicles (P = 0.07) compared with that of the eCG group, whereas secondary and antral follicular populations were similar. Rates of late atretic follicles assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique were not different between treatments, but the eCG group showed a significantly higher number of mid-atretic follicles compared with that of the Bio group. Nuclear in vitro oocyte maturation (IVM), measured as metaphase II rate, and in vitro steroidogenic response of cumulus-oocyte complexes, measured by ELISA, did not show significant differences between treatments. However, confocal study showed that cytoplasmic maturation of oocytes, in terms of cortical granule migration rate, was significantly higher in the Bio group compared with that after the eCG treatment. In conclusion, transient doe-litter separation seems to improve ovarian response in terms of follicular health and oocyte competence compared with that after the eCG treatment. Therefore, a 24-h-long transient weaning could be an alternative nonhormonal method for synchronizing estrus in primiparous lactating rabbit does inseminated in the early postpartum period.     

Effect of superovulation induction on embryonic development on day 5 and subsequent development and survival after nonsurgical embryo transfer in pigs

To evaluate the effects of eCG dosage on recovery and quality of Day 5 embryos and on subsequent development and survival after embryo transfer, batches of 5 to 10 donor sows were treated with 1000 or 1500 IU eCG. Recipients from the same batch were synchronously treated with 800 IU eCG. Ovulation was induced with 750 IU hCG (72 h after eCG) in donors and recipients. Donors were inseminated and embryos were collected at 162 h after hCG (120 h after ovulation). Ovulation rate was lower using 1000 IU eCG (28.5+/-11.7; n=48) than 1500 IU eCG (45.7+/-20.3; n=32; P<0.0001). Embryo recovery rate (82.9+/-16.9%) and percentage expanded blastocysts (56.2+/-31.4%) were similar (P>0.05). Expanded blastocysts from each group of sows were pooled into 2 groups within eCG treatment, containing embryos from normally ovulating sows (< or = 25 corpora lutea [CL]) or from superovulated sows (> 25 CL). Average diameter and number of cells of a random sample of the expanded blastocysts per pool were recorded. The average diameter of blastocysts (160.5+/-11.5 microm) was not affected by eCG dosage or ovulation rate (P>0.10). The average number of cells per embryo was higher in the 1000 IU eCG group (84.3+/-15.3) than in the 1500 IU eCG group (70.2+/-1.9; P<0.05) but was similar for normal and superovulated donors within each eCG group (P>0.10). Of the 4 groups, litters of 28 to 30 blastocysts were nonsurgically transferred to 27 synchronous recipients. Pregnant recipients were slaughtered on Day 37 after hCG treatment to evaluate embryonic development and survival. Pregnancy rate for the 1000 and 1500 IU eCG donor groups was 71% (10/14) and 46% (6/13; P>0.10), respectively. The number of implantations and fetuses for the 1000 IU eCG groups was 12.9+/-3.0 and 11.1+/-2.7, and 14.2+/-7.0 and 10.5+/-4.6, respectively, for the 1500 IU eCG groups (P>0.10). After post-priory categorizing the litters of blastocysts to below or above the average diameter (158 microm) of the transferred embryos, irrespective of eCG dosage or ovulation rate, the pregnancy rate was 43% (6/14) and 77% (10/13; P<0.10), respectively. Post-priory categorizing the transferred litters to below or above the average number of cells per embryo litter, irrespective of eCG dosage or ovulation rate, showed no differences in pregnancy rates or number of implantations and fetuses (P>0.10). It was concluded that eCG dosage affects embryonic development at Day 7 after hCG, and this effect was not due to ovulation rate. Embryonic survival after nonsurgical transfer was not related to eCG dosage but tended to be related to the diameter of the blastocysts.     

Progesterone treatment, FSH plus eCG, GnRH administration, and Day 0 Protocol for MOET programs in sheep

The effect of various superstimulatory treatments on the number of corpora lutea, fertilization rate, and embryo yield was studied in sheep. Overall, data from 708 Merino donors and 4262 embryos were analyzed in four experiments. In Experiment 1, varying intervals of progesterone treatment (5 to 14 d) before follicle-stimulating hormone (FSH) administration did not significantly affect the proportion of responding donors, the mean number of corpora lutea, or the mean number of recovered and transferable embryos per donor. In Experiment 2, a single dose of equine chorionic gonadotropin (eCG, 200 or 300 IU) combined with the FSH treatment (i.e., given at CIDR removal) reduced the number and the quality of embryos compared with that for not giving eCG (P < 0.05). In Experiment 3, one dose of gonadotropin-releasing hormone (GnRH) given 24 h after CIDR removal improved the number of transferable embryos compared with that for not giving GnRH (P < 0.05). In Experiment 4, the new superstimulatory Day 0 Protocol, which includes starting FSH treatment at the emergence of Wave 1 (i.e., soon after ovulation, in the absence of a large follicle), improved ovarian response, with a tendency to produce more embryos compared with that for the Traditional Protocol. In summary, this study, analyzing data from various pharmacologic treatments, allows an improvement from four to eight transferable embryos per treated donor in multiple ovulation and embryo transfer programs in sheep.     

Effects of recombinant human follicle-stimulating hormone on embryo development in mice

We have developed a protocol using recombinant human follicle-stimulating hormone (rhFSH) to induce ovarian stimulation in the mouse to investigate its impact on preimplantation embryo development. Embryos were collected from adult female C57Bl/6 x CBA F1 mice treated with rhFSH (0, 2.5, 5.0, 10.0, or 20.0 IU) or 5 IU equine chorionic gonadotropin (eCG). Embryos were also recovered from nontreated control mice. Embryos were cultured in vitro for 88 h, and the stage of development was morphologically assessed. The allocation of cells to the inner cell mass or trophectoderm of blastocysts was determined by differential nuclear staining. The expression of insulin-like growth factor 2 (IGF-II), the insulin-like growth factor receptor (IGF-II receptor), and vascular endothelial growth factor (VEGF) in blastocysts was measured by real-time RT-PCR. Blastocyst development was reduced in the 10 (72.3 +/- 5.1%) and 20 (77.3 +/- 5.6%) IU rhFSH groups compared with control embryos (96.7 +/- 1.0%). The number of inner cell mass cells was reduced (P < 0.001) in the 5, 10, and 20 IU rhFSH groups and the eCG group compared with control embryos. We did not find any effect of rhFSH treatment on IGF-II, IGF-II receptor, or VEGF expression in blastocysts compared with the control group. eCG treatment, however, significantly increased the expression of IGF-II in blastocysts. These results indicate that ovarian stimulation with rhFSH impairs the in vitro development of preimplantation mouse embryos, and these results may have potential implications for clinical ovarian stimulation during infertility treatment and subsequent embryo quality.     

Optimization of cryopreservation of buffalo (Bubalus bubalis) blastocysts produced by in vitro fertilization and somatic cell nuclear transfer

The objective of this study was to optimize cryopreservation conditions for buffalo in vitro produced (IVP) embryos. The in vitro fertilized (IVF) and somatic cell nuclear transferred (SCNT) blastocysts were vitrified with either 40% ethylene glycol (EG), 25% EG + 25% dimethylsulfoxide (DMSO), or 20% EG + 20% DMSO + 0.5 m sucrose, and the IVF blastocysts produced from abattoir-derived ovaries were also slow-frozen with either 10% EG or 0.05 m trehalose dehydrate + 1.8% EG + 0.4% BSA. Cryosurvival rates of blastocysts harvested on various days or at various developmental stages were also examined. In this study: (1) vitrification with 20% EG + 20% DMSO + 0.5 m sucrose had the best cryopreservation efficiency; (2) IVF and SCNT blastocysts had similar cryotolerance (P > 0.05); (3) after thawing, slow-frozen blastocysts reexpanded earlier than the vitrified blastocysts (P < 0.01); (4) cryosurvival rate of expanded blastocysts was higher than that of early blastocysts (P < 0.05); (5) cryosurvival rates of Days 5 to 7 blastocysts (Day 0 = day of IVF or SCNT) were higher than those of Days 8 to 9 blastocysts (P < 0.01); and (6) after embryo transfer, pregnancy rates for fresh and cryopreserved blastocysts were not different (P > 0.05). In conclusion, vitrification of Days 6 to 7 expanded blastocysts with 20% EG + 20% DMSO + 0.5 m sucrose was optimal for cryopreservation of buffalo IVP embryos.     

Isolation and initial culture of porcine inner cell masses derived from in vitro-produced blastocysts

The present study was conducted to isolate and culture inner cell mass (ICM) primarily derived from in vitro-produced blastocysts and to develop the culture conditions for the ICM cells. In Experiment 1, immunosurgically isolated ICMs of blastocysts derived from in vitro fertilization (IVF), somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) were seeded onto STO cells. Primary colonies from each isolated ICM were formed with a ratio of 28.9, 30.0 and 4.9%, respectively. In Experiment 2, blastocysts collected from IVF were directly seeded onto a feeder layer with or without zona pellucida (ZP), or were subjected to ICM isolation by immunosurgery. Primary colonies were formed in 36.8% of isolated ICMs and 19.4% in intact blastocysts without ZP. In Experiment 3, ICMs from IVF blastocysts were seeded onto STO cells, mouse embryonic fibroblast (MEF) or porcine uterine epithelial cells (PUEC). On STO and MEF cells, 34.5 and 22.2% of primary colonies were formed, respectively. However, no primary colony was formed on the PUEC or in feeder-free condition. In Experiment 4, ICMs from IVF blastocysts were cultured in DMEM + Ham's F10 (D/H medium), DMEM + NCSU-23 (D/N medium) or DMEM alone. When D/H medium or D/N medium was used, 21.7 or 44.4% of primary colony were formed, respectively, while no primary colony was formed in DMEM alone. These cells showed alkaline phosphatase activity and could be maintained for up to five passages. In suspension culture, cells formed embryoid bodies. These results demonstrate that porcine ICM could be isolated and cultured primarily from in vitro-produced blastocysts with a suitable culture system.     

Quantifying difference in gene expression profile between bovine blastocysts derived by in vitro fertilization and somatic cell nuclear transfer

Epigenetic reprogramming intensely occurs in somatic-cell nuclear transfer (SCNT) embryos, which highlights the importance of proper expressions of reprogramming-related genes in SCNT embryos. We here assessed gene expression profiles (GEPs) difference between bovine blastocyst groups derived by in-vitro fertilization (IVF) or SCNT; in SCNT, cumulus cells and ear skin fibroblasts were used for cSCNT and fSCNT blastocysts, respectively. We obtained GEPs of 15 reprogramming-related genes in single blastocysts using multiplex PCR and found a broad range of variations in their GEPs. Weighted root-mean-square deviation (wRMSD) analysis, which calculates the deviation of SCNT blastocysts' GEPs from IVF blastocysts' mean GEP, found a significant difference between IVF and fSCNT and between cSCNT and fSCNT blastocysts (p < 0.001) but not between IVF and cSCNT. Since the fibroblasts' GEP was more distant from the IVF blastocysts' than the cumulus cells', it might partly explain the less similarity of fSCNT blastocysts' GEPs to the IVF's mean GEP. Our wRMSD method succeeds in expressing in figures how different two comparable embryo groups of different derivations are in GEP, which would be useful to select a better embryo derivation protocol among the candidates prior to field applications.     

The effect of addition of equine chorionic gonadotropin to a progesterone-based estrous synchronization protocol in buffaloes (Bubalus bubalis) under tropical conditions

Poor estrus expression and anestrus decrease the reproductive efficiency of buffaloes. The objective of this study was to determine whether the addition of equine chorionic gonadotropin (eCG) to an estrous synchronization protocol and timed insemination could improve ovulation and pregnancy rates of anestrous buffalo cows under tropical conditions. The study population comprised 65 lactating Murrah buffalo cows which were assigned to CIDR (n = 33) or CIDR + eCG (n = 32) treatment groups. Cows in the CIDR group were fitted for 8 d with a controlled intravaginal drug release (CIDR) device containing 1.38 g progesterone, received GnRH (10 μg i.m.) on D 0, PGF_2α (750 μg i.m.) on D 7, and GnRH (10 μg i.m.) on D 9; whereas cows in the CIDR + eCG group received the same treatment plus eCG (500 IU, i.m.) at the time of PGF_2α treatment. All cows were inseminated 16-20 h after the second GnRH treatment. Blood samples were obtained 10 d before the start of synchronization treatment (Day -10) and at the onset of treatment (Day 0). Cows with plasma progesterone concentrations <1 ng/mL recorded in both samples (Low-Low levels of P4) were classified as non-cyclic cows. Similarly, when either one or both of the sample pair contained concentrations of serum progesterone ≥1 ng/mL (High-High, Low-High, or High-Low levels of P4), the buffaloes were classified as cyclic cows. Ovulation rate, defined as the number of buffaloes with at least one corpus luteum 10 days after insemination, was significantly higher (P = 0.018) in the CIDR + eCG (84.4%) cows than in the CIDR cows (57.6%). Pregnancy rate was numerically lower in CIDR (27.3%) than CIDR + eCG (40.6%) cows, though differences were not significant (P = 0.25). Pregnancy rates for CIDR + eCG cows were similar to that of cows inseminated after natural estrus (40.9%; 29/71). In the non-cyclic animals, higher ovulation rates (P = 0.026) were recorded for the CIDR + eCG (81%) than for the CIDR cows (47.4%). Our results indicate that the addition of eCG to a progesterone-based estrous synchronization regimen substantially improves the ovulation rate in non-cyclic buffaloes. When this treatment is followed by timed AI, pregnancy rates achieved in anestrous buffaloes, whether cyclic and non-cyclic, may approach the rates observed in cows inseminated at natural estrus.     

Expression of leptin ligand and receptor and effect of exogenous leptin supplement on in vitro development of porcine embryos

The present study investigated the expression of ligand and receptor for leptin, and the effect of leptin supplementation on preimplantation development of porcine in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos. The IVF embryos were produced using frozen boar semen and SCNT embryos were obtained by nuclear transfer of fetal fibroblasts into enucleated oocytes. The protein expression of leptin ligand and receptor was investigated in in vitro matured oocytes, 2-, 4- and 8-cell embryos, morulae and blastocysts derived from IVF and SCNT using immunofluorescence. Both the ligand and receptor were detected in in vitro matured oocytes and all stage of IVF and SCNT embryos. The IVF and SCNT embryos were cultured in modified North Carolina State University (mNCSU)-23 medium supplemented with various concentrations (0, 1, 10, 100 or 1000 ng/mL) of leptin. The rates of cleavage at day 2 and blastocyst formation at day 7, and cell number of blastocysts were monitored as experimental parameters. In SCNT embryos, supplementing with 1000 ng/mL leptin significantly (P<0.05) increased the rate of blastocysts formation (20.2% versus 12.9%) and total cell number (54.6+/-17.4 versus 45.1+/-15.2) compared to the control group. In IVF embryos, leptin supplementation did not affect preimplantation embryo development and cell number in blastocysts. In conclusion, the present study demonstrated the expression of leptin ligand and receptor and the embryotropic effect of leptin in SCNT embryos.     

Development of a modified straw method for vitrification of in vitro-produced bovine blastocysts and various genes expression in between the methods

This study evaluated a modified plastic straw loading method for vitrification of in vitro-produced bovine blastocysts. A modified straw was used with a depressed area on its inner surface to which embryos attach. In vitro-produced blastocysts were randomly assigned into three groups: (i) blastocysts attached to the inner surface of a plastic straw (aV), (ii) blastocysts attached to the inner surface of a modified plastic straw (maV), and (iii) non-vitrified blastocysts (control). The recovery rates were not significantly different between aV and maV groups (95.8% vs. 94.3%). The post-thaw survival rate did not significantly differ between aV and maV groups (86.4% vs. 88.2%). The total cell numbers of blastocyst was higher in control than in aV and maV groups (142 ± 21.8 vs. 117 ± 29.7 and 120 ± 25.2; P < 0.05), but not significantly differ between aV and maV groups. The mRNA levels of pro-apoptosis related genes Bax and Caspase-3 were higher in aV and maV than in control (P < 0.05). By contrast, the mRNA levels of anti-apoptotic genes Bcl-2 and Mcl-1 and of antioxidant-related genes MnSOD and Prdx5 were lower in aV and maV than in control (P < 0.05). Confocal microscopy analysis of Golgi apparatus and mitochondria showed that the fluorescence intensity of Golgi apparatus and mitochondria was higher in control than in aV and maV groups. In conclusion, both aV and maV methods can be used to successfully vitrify IVP blastocysts, with maV method to be preferable because of its easiness in embryo loading.     

The effects of superovulation of donor sows on ovarian response and embryo development after nonsurgical deep-uterine embryo transfer

This study aimed to evaluate the effectiveness of superovulation protocols in improving the efficiency of embryo donors for porcine nonsurgical deep-uterine (NsDU) embryo transfer (ET) programs. After weaning (24 hours), purebred Duroc sows (2–6 parity) were treated with 1000 IU (n = 27) or 1500 IU (n = 27) of eCG. Only sows with clear signs of estrus 4 to 72 hours after eCG administration were treated with 750 IU hCG at the onset of estrus. Nonhormonally treated postweaning estrus sows (n = 36) were used as a control. Sows were inseminated and subjected to laparotomy on Days 5 to 6 (Day 0 = onset of estrus). Three sows (11.1%) treated with the highest dosage of eCG presented with polycystic ovaries without signs of ovulation. The remaining sows from nonsuperovulated and superovulated groups were all pregnant, with no differences in fertilization rates among groups. The number of CLs and viable embryos was higher (P < 0.05) in the superovulated groups compared with the controls and increased (P < 0.05) with increasing doses of eCG. There were no differences among groups in the number of oocytes and/or degenerated embryos. The number of transferable embryos (morulae and unhatched blastocysts) obtained in pregnant sows was higher (P < 0.05) in the superovulated groups than in the control group. In all groups, there was a significant correlation between the number of CLs and the number of viable and transferable embryos, but the number of CLs and the number of oocytes and/or degenerated embryos were not correlated. A total of 46 NsDU ETs were performed in nonhormonally treated recipient sows, with embryos (30 embryos per transfer) recovered from the 1000-IU eCG, 1500-IU eCG, and control groups. In total, pregnancy and farrowing rates were 75.1% and 73.2%, respectively, with a litter size of 9.4 ± 0.6 piglets born, of which 8.8 ± 0.5 were born alive. There were no differences for any of the reproductive parameters evaluated among groups. In conclusion, our results demonstrated the efficiency of eCG superovulation treatments in decreasing the donor-to-recipient ratio. Compared with nonsuperovulated sows, the number of transferable embryos was increased in superovulated sows without affecting their quality and in vivo capacity to develop to term after transfer. The results from this study also demonstrate the effectiveness of the NsDU ET procedure used, making possible the commercial use of ET technology by the pig industry.     

Expression patterns of sirtuin genes in porcine preimplantation embryos and effects of sirtuin inhibitors on in vitro embryonic development after parthenogenetic activation and in vitro fertilization

We examined the expression patterns of porcine sirtuin 1 to 3 (Sirt1-3) genes in preimplantation embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). We also investigated the effects of sirtuin inhibitors (5 mM nicotinamide [NAM] and 100 μM sirtinol) on embryonic development of PA and IVF embryos under in vitro culture (IVC). The expression patterns of Sirt1-3 mRNA in preimplantation embryos of PA, IVF, and SCNT were significantly (P < 0.05) decreased from metaphase stage of oocyte to blastocyst stage. Especially, the expressions of Sirt1-3 in SCNT blastocysts were significantly (P < 0.05) lower and Sirt2 in PA blastocyst was significantly higher compared with the IVF blastocysts. Treatment with sirtuin inhibitors during IVC resulted in significantly (P < 0.05) decreased blastocyst formation and total cell number of blastocyst derived from PA (NAM: 29.4% and 29.6, sirtinol: 31.0% and 30.3, and control: 40.9% and 41.7, respectively) and IVF embryos (NAM: 10.4% and 30.9, sirtinol: 6.3% and 30.5, and control: 16.7% and 42.8, respectively). There was no significant difference in cleavage rate in both PA and IVF embryos. The early and expanded blastocyst formations at Day 7 were significantly lower in the sirtuin inhibitors-treated groups than the control. It was demonstrated that sirtuin inhibitor (NAM) influenced the percentage of blastocyst formation and total cell number of PA derived blastocyst when NAM was added during Day 4 to 7 (22.1% and 32.4) or Day 0 to 7 (23.1% and 31.6) of IVC compared with the control (41.8% and 41.5). No significant difference in cleavage rates appeared among the groups. The blastocysts derived from PA embryos treated with sirtuin inhibitors showed lower (P < 0.05) expressions of POU5F1 and Cdx2 genes. Also, Sirt2 mRNA expression was significantly decreased in sirtinol treated group and Sirt3 mRNA expression was also significantly decreased in both NAM and sirtinol treated groups compared with the control. In conclusion, these results suggest that sirtuins may have a physiological and important role in embryonic development of porcine preimplantation embryos by regulating essential gene expressions of developing embryos. These findings could have implications for understanding the role of sirtuins during embryo development and for improving SCNT and related techniques.     

Effects of progesterone presynchronization and eCG on pregnancy rates to GnRH-based, timed-AI in beef cattle

Three experiments were conducted to determine the effects of low-dose progesterone presynchronization and eCG on pregnancy rates to GnRH-based, timed-AI (TAI) in beef cattle (GnRH on Day 0, PGF_2α on Day 7, with GnRH and TAI on Day 9, 54-56 h after PGF_2α). Experiments 1 and 2 were 2 × 2 factorials with presynchronization (with or without a once-used CIDR; Days −15 to 0 in Experiment 1 and Days −7 to 0, with PGF_2α at insertion, in Experiment 2), and with or without 400 IU eCG on Day 7 in suckled cows. In Experiment 3, suckled cows and nulliparous heifers were either presynchronized with a twice-used CIDR (Days −5 to 0) and PGF_2α at insertion, or no treatment prior to insertion of a new CIDR (Days 0-7). Presynchronization increased (P < 0.05) ovulation rate to GnRH on Day 0 (75.0% vs 48.7%, 76.7% vs 55.0%, and 60.0% vs 36.1% for Experiments 1, 2, and 3, respectively), increased the diameter of the preovulatory follicle in Experiments 1 and 2, and increased the response to PGF_2α (regardless of parity) in Experiment 1 (P < 0.01), and in primiparous cows in Experiment 2 (P < 0.01). Effects of presynchronization on pregnancy rates (53.4% vs 54.1%, 57.7% vs 45.3%, and 54.3% vs 44.4% for Experiments 1, 2, and 3, respectively) were influenced by parity and eCG (P < 0.05). Treatment with eCG had no effect (P > 0.05) on the diameter of the preovulatory follicle (Experiment 1), or the response to PGF_2α (Experiments 1 and 2), but tended (P = 0.08) to improve pregnancy rates, especially in primiparous cows that were not presynchronized (P < 0.01). However, the effects of eCG and presynchronization were not additive.