Progestogen supplementation during superovulation leads to higher embryo viability and TGFB1 gene expression in sheep.

This study aimed to compare the effect of the administration of either medroxyprogesterone acetate (MPA) or progesterone (P4) in superovulation (SOV) treatments applied during the first follicular wave on follicular development, embryo yield, and the expression of genes related to pluripotency maintenance, differentiation of the trophectoderm, cell growth and differentiation, apoptosis and energy metabolism in sheep embryos. The estrous cycle of 36 multiparous ewes was synchronized with a short protocol, and the animals were randomly allocated to three groups. At the beginning of SOV, 12 ewes per treatment received an intravaginal sponge impregnated with 60 mg of MPA (TMPA), or an intravaginal device containing 0.33 g of P4 (TP4), or received no progestogen treatment (CON). The device was kept until the fifth dose of FSH. Ewes were mated with five fertile rams. Gene expression was performed by RT-qPCR using grade I and II blastocysts. The numbers of corpora lutea, total structures and viable embryos recovered per ewe were similar (P > 0.05) among groups. However, the viability rate was higher in TP4 (71.9 ± 16.3%) compared to CON (24.4 ± 16.8%; P = 0.01) and similar to TMPA (49.9 ± 16.3%; P = 0.2). Similarly, when compared with CON, treatment with P4 or MPA positively regulated the TGFB1 transcript involved in cell proliferation and differentiation (P = 0.01 and P = 0.03, respectively). In conclusion, supplementation with P4 during the first follicular wave of the estrous cycle improves embryo viability and alters the expression of the TGFB1 gene.

Superovulatory response and embryo quality in Boer does following dietary supplementation with different sources of omega-3 and omega-6 fatty acids during the breeding season.

The purpose of this study was to determine effects of various sources of omega-3 and omega-6 fatty acids on ovarian response and embryo quality in Boer does when there was a superovulation treatment regimen imposed. Pluriparous does were randomly assigned to be treated with 300 g of one of four experimental supplements containing linseed oil (LO), soybean oil (SO), palm oil (PO), or a control supplement without fatty acids (CO), for 15 days. Does were fitted with a controlled internal drug release (CIDR) device containing 0.3 g progesterone for 7 days. At 48 h before CIDR withdrawal, does were treated with 80 mg follicle-stimulating hormone (FSH) administered at 12 h intervals. Embryos were collected 7 days after the last natural mating. Estrous response and interval between CIDR withdrawals to estrous onset were similar between treatments (P > 0.05). Number of ovulations was similar for does in the different groups (10.0, 9.2, 7.0, and 7.0, in LO, SO, PO, and CO, respectively; P > 0.05). There was premature luteal regression in does of the SO, PO, and CO groups, except in LO group. The LO-treated does had a larger (P < 0.05) mean number of ova/embryos recovered than does of SO, PO, and CO groups (7.2, 2.0, 0.2, 0.2, respectively) and transferable embryos (5.1, 1.4, 0.2, 0.2, respectively). These results indicate that including LO in supplements may be a feasible strategy for preventing premature luteal regression and improving embryo quality in goats treated to induce follicular super-stimulation for induction of superovulation.

Effects of feeding OmniGen-AF® on superovulatory response in donor beef cows: I. Serum progesterone and cortisol, embryo recovery and quality.

Optimal results in cattle embryo transfer are limited by the variation in ova recovery, fertilization rate and embryo quality experienced with superovulation. Inflammation and immune dysregulation may be contributing factors. This study, evaluated feeding OmniGen-AF® (OG), a nutritional supplement that reduces inflammation and supports immune health, on superovulatory response and serum progesterone and cortisol concentrations in embryo donors treated with two different doses of Folltropin®-V (FSH). Angus cross-bred beef cows (n = 24) were assigned to four groups, fed OG at 0 or 56 g/animal/day for 49 days and were treated with 200 or 400 mg FSH to induce superovulation. Treatments for superovulation started after feeding OG for 28 days and ova were non-surgically recovered 7 days after estrus and graded for quality. More transferrable embryos (P < 0.05) were recovered from cows fed 56 g OG and treated with 400 compared with 200 mg FSH. Percent degenerate embryos recovered from cows treated with the 400 mg FSH dose was threefold greater (P < 0.05) when fed no OG compared with 56 g OG. Serum progesterone on day of embryo collection was greater (P < 0.05) in OG-supplemented cows and cows treated with 200 mg FSH. Serum cortisol was not affected (P > 0.10) by FSH dose or OG-feeding, but was greatest (P <  0.05) on Days 0 and 42 of the feeding period. In summary, the improvement in embryo quality with OG-feeding may relate to a greater serum progesterone concentration.

Annatto (Bixa orellana) δ-TCT Supplementation Protection against Embryonic Malformations through Alterations in PI3K/Akt-Cyclin D1 Pathway.

Protective action by annatto-derived delta-tocotrienol (δ-TCT) and soy-derived alpha-tocopherol (α-TOC) through the regulation of the PI3K/Akt-cyclin D1 pathway against nicotine-induced DNA damage is the focus of the present study. Nicotine, which has been widely reported to have numerous adverse effects on the reproductive system, was used as a reproductive toxicant. 48 female balb/c mice (6⁻8 weeks) (23⁻25 g) were randomly divided into eight groups (Grp.1⁻Grp.8; n = 6) and treated with either nicotine or/and annatto δ-TCT/soy α-TOC for seven consecutive days. On Day 8, the females were superovulated and mated before euthanization for embryo collection (46 h post-coitum). Fifty 2-cell embryos from each group were used in gene expression analysis using Affymetrix QuantiGene Plex2.0 assay. Findings indicated that nicotine (Grp.2) significantly decreased (p < 0.05) the number of produced 2-cell embryos compared to the control (Grp.1). Intervention with mixed annatto δ-TCT (Grp.3) and pure annatto δ-TCT (Grp.4) significantly increased the number of produced 2-cell embryos by 127% and 79%, respectively compared to Grp.2, but these were lower than Grp.1. Concurrent treatment with soy α-TOC (Grp.5) decreased embryo production by 7%. Supplementations with δ-TCT and α-TOC alone (Grp.6-Grp.8) significantly increased (p < 0.05) the number of produced 2-cell embryos by 50%, 36%, and 41%, respectively, compared to control (Grp.1). These results were found to be associated with alterations in the PI3K/Akt-Cyclin D1 genes expressions, indicating the inhibitory effects of annatto δ-TCT and soy α-TOC against nicotinic embryonic damage. To our knowledge, this is the first attempt in studying the benefits of annatto δ-TCT on murine preimplantation 2-cell embryos.

[Intervention of controlled ovarian hyperstimulation cycle by Chinese medical herbs for Shen tonifying, blood nourishing and activating: a randomized clinical trial].

OBJECTIVE: To study the effect of Chinese medical herbs for Shen tonifying, blood nourishing and activating (CMHSTBNA) on the cycle of controlled ovarian hyperstimulation (COH) of assisted reproductive technique (ART). METHODS: A large sample randomized control trial was performed. Infertility women patients, younger than 42 years (infertility due to tubal factor and/or male factor) were randomly assigned to the CMHSTBNA intervention group (abbreviated as the treated group) and the control group, 184 cases in each group. All underwent COH. Those in the treated group received assist therapy of CMHSTBNA from the menstrual period day 2 -3 of COH to the day of oocytes retrieved. The serum hormone level [including estrogen (E2), progesterone(P), luteal hormone (LH) on the day of human chorionic gonadotrophin (hCG) administration], the medication days and dosage of gonadotropin (Gn), the number of oocytes retrieved, the fertilization rate, and the good-quality embryo rate were observed and compared with the control group. RESULTS: The endometrial thickness on the day of oocytes retrieved was 10.85 +/- 1.63 mm in the treated group, larger than that in the control group (10.50 +/- 1.49 mm) (P <0.05). The good-quality embryo rate and the frozen rate were 48. 9% and 39. 7% respectively in the treated group, superior to those of the control group (45. 4% and 35. 8% respectively), showing statistical difference (P < 0.05). On the day of hCG administration, favorable tendency was shown in the serum levels of estradial (E2), progesterone (P), luteinizing hormone (LH), the medication days and dosage of Gn, the number of oocytes retrieved, the fertilization rate, and the cleavage rate, showing no statistical difference when compared with the control group (P >0.05). CONCLUSION: The combined application of CMHSTBNA and gonado-trophic hormones in COH cycle could elevate the embryo quality, improve the endometrial state, thus laying foundation for successful in vitro fertilization/intracytoplasmic sperm embryo transfer.

The role of nutritional supplementation on the outcome of superovulation in cattle.

Since the 1990s nutritional supplements including protein, fatty acids, vitamins, and minerals have been used to try and improve the superovulatory response of embryo donors in cattle. However, the accumulated information indicates that nutritional supplementation with protein, fatty acids, or minerals does not increase the number of viable embryos from superovulated cattle. Most of the evidence has shown that vitamin supplementation may increase the mean production of transferable embryos, but only in cows, as a detrimental effect on embryo viability has been reported in young heifers. Nevertheless, vitamin supplementation seems to be effective only when compared with control cows displaying a poor mean embryo production (i.e. less than four viable embryos), questioning the economical significance of such approach. Detrimental effects on embryo development have been reported in superovulated cattle supplemented with protein or fatty acids as well. New approaches to investigate the role of nutritional supplementation on superovulatory outcome in cattle are suggested in the present review. Overall, the available evidence indicates that nutritional supplementation strategies tested are not an effective approach to enhance the superovulatory outcome of well-fed cattle donors.

Effect of estradiol benzoate used at the start of a progestagen treatment on superovulatory response and embryo yield in lactating and non-lactating llamas.

Two experiments were conducted to determine the effect of estradiol benzoate (EB) and intravaginal progestagen treatment on ovarian follicular dynamics and superstimulatory response in eCG-treated llamas. The purpose of Experiment 1 was to evaluate the effect of EB and progestagen treatment starting at different phases of dominant follicle (DF1) development on regression pattern and subsequent follicle wave emergence (WE2) in lactating and non-lactating llamas. Early lactating (n=24, 30+/-4 days postpartum) and non-lactating (n=24) females were assigned in equal numbers (n=8) to one of three groups according to the phase of DF1 (growing, static or regressing) determined by ultrasonography from day -3 to day 0. At day 0, llamas received an intravaginal sponge (MPA, 150 mg) and 5mg of MPA (i.m.). Half of the females (n=4) of each group were injected with 2mg (i.m.) of EB and half were not (control group). A 2 x 2 x 3 (lactational status, EB treatment and follicular phases) factorial design was used. Each sponge was removed 8 days later. Ovaries were monitored from day 0 to day 12. Daily blood samples were taken to determine 17beta-estradiol (E(2)) profiles from day 0 to day 8. The DF1 regression pattern was not affected (P>0.05) by the phase of follicle wave at the start of the treatment or any interactions among main effects. Follicle wave emergence in EB-treated llamas was delayed (P<0.05) by 2.3 days compared with non-treated llamas. Following EB treatment, plasma concentrations of E(2) were greater (P<0.05) from day 1 to day 5 in the treated than in non-treated females, but not from day 6 onward (P>0.05). Experiment 2 was designed to evaluate the effect of this treatment on the ovarian superovulatory response and embryo yield following eCG treatment administered on day of follicular wave emergence as determined in the Experiment 1. The same lactating (n=18, 61+/-4 days postpartum) and non-lactating (n=18) llamas at random stages of follicle wave were treated as those in Experiment 1 and received 1200IU of eCG at the time of WE2 (EB-treated=day 6.5 and non-treated=day 4.5). Llamas were mated 5 days after sponge withdrawal. A second mating was allowed 24h later. Embryos were collected between 7 and 8 days after the first mating and blood samples were taken to determine progesterone (P(4)) concentrations. The mean number of follicles on day of mating and the number of CL on day of embryo collection were not affected by lactational status, EB treatment or their interactions (P>0.05). Ovulation rate and mean (+/-SEM) number of recovered embryos for EB treatment group (67.5% and 2.4+/-0.4) were greater (P<0.05) than for no EB treatment (51.1% and 1.1+/-0.4). Plasma P(4) concentrations and number of CL per llama were correlated (r=0.49; P=0.014). In conclusion, progestagen plus EB treatment facilitates the prediction of the emergence of a new follicular wave approximately 6 days after treatment and resulted in a higher ovulation rate and embryo production in ovarian superstimulated llamas regardless of lactational status.

Glutathione content and antioxidant enzyme expression of in vivo matured sheep oocytes.

Retinoids play important roles in many diverse biological functions such as cell growth, morphogenesis, differentiation, and reproduction. Previous studies demonstrated that retinol administration to ewes, followed by natural service, resulted in embryos with improved competence to develop under standard in vitro conditions (5% CO(2) in air). Additional studies provided evidence that retinol may have some antioxidant effect by improving blastocyst development in cattle under atmospheric conditions (5% CO(2) in air). Glutathione is an important non-protein, sulphydryl compound found in oocytes and embryos, which acts to decrease oxidative stress. The purpose of the present study was to evaluate the effects of retinol administration to ewes on the content of glutathione and glutathione-related and antioxidant enzymes in in vivo matured sheep oocytes. Briefly, ewes were administered retinol or vehicle during superovulation, and after 60h the oviducts were removed and mature oocytes collected. Glutathione content did not differ significantly between oocytes collected from retinol-treated ewes (6.78+/-3.81pmol/oocyte) and control ewes (6.38+/-1.58pmol/oocyte). Transcripts encoding for manganese superoxide dismutase (Mn-SOD), copper zinc superoxide dismutase (Cu-Zn SOD), glutathione synthetase (GS), and glutathione transferase pi (GSTp) were detected in single ovine oocytes; however, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis did not reveal any significant differences in transcripts between oocytes from retinol-treated ewes and those from control ewes.

Embryo yield and quality following dietary supplementation of beef heifers with n-3 polyunsaturated fatty acids (PUFA).

The objective of this study was to examine the effects of dietary n-3 polyunsaturated fatty acid (n-3 PUFA) supplementation on embryo yield and quality in heifers. Animals were individually offered barley straw and concentrate diets supplemented with either palmitic acid (C16:0; CON) or a partially rumen protected n-3 PUFA-enriched supplement. Following oestrous cycle synchronisation, superovulation was induced using FSH. Blood samples were collected for the measurement of fatty acids, metabolites, insulin and IGF-1. On day 7 post-insemination the number of ovulations was estimated and embryos recovered non-surgically and quality graded. At embryo recovery 50 ml of the uterine flushing was collected from each horn for fatty acid analysis. Grade 1 embryos were isolated, snap frozen in liquid nitrogen and stored at -80 degrees C. mRNA expression for six genes, LIF, BAX, Cx43 and E-CAD associated with embryo development, and PPAR-alpha and -delta, associated with lipid metabolism was analysed. The n-3 PUFA supplementation increased plasma n-3 PUFA concentration (P<0.05) and reduced n-6:n-3 PUFA ratio (P<0.05). Uterine concentration of the n-3 PUFA, eicosapentaenoic acid was increased (P<0.05) and the concentration of arachidonic acid decreased (P<0.05) following n-3 PUFA supplementation. While CON increased triglyceride concentrations, diet did not affect the other plasma metabolites, insulin or IGF-1 (P>0.05). Similarly, there was no effect of diet on superovulation rate, embryo recovery rate, embryo quality (P>0.05) or mRNA expression of the genes examined (P>0.05).

Sodium nitroprusside treatment during the superovulation process improves ovarian response and ovarian expression of vascular endothelial growth factor in aged female mice.

OBJECTIVE: To investigate whether sodium nitroprusside (SNP) treatment during the superovulation process improves ovarian response and oocyte developmental competence in aged female mice. DESIGN: Controlled experimental study. SETTING: Large urban medical center. ANIMAL(S): C57BL inbred female mice of three age groups: 6 to 9, 14 to 16, and 25 to 27 weeks. INTERVENTION(S): Female mice were co-injected intraperitoneally with SNP (1 muM or 10 muM) and pregnant mare's serum gonadotropin (PMSG), followed by human chorionic gonadotropin injection 48 hours later and then mated with individual males. After 18 hours, zygotes were flushed and the ovaries were isolated. The control group was injected with PMSG. MAIN OUTCOME MEASURE(S): The number of zygotes flushed, embryo development to blastocyst stage, and vascular endothelial growth factor (VEGF) expression in ovary. RESULT(S): Treatment with SNP statistically significantly increased the number of flushed zygotes and blastocyst formation rate in mice aged 25 to 27 weeks, not but in mice aged less than 16 weeks compared with the control group. The SNP treatment in aged mice increased VEGF expression of the ovary in a dose-dependent manner. CONCLUSION(S): These results demonstrate that SNP treatment during the superovulation process improves ovarian response and oocyte developmental competence in aged female. The positive effect of SNP may be associated with increased VEGF expression.

Embryo production and quality of Holstein heifers and cows supplemented with beta-carotene and tocopherol.

The hypothesis was that the intramuscular injection (i.m.) of beta-carotene associated to tocopherol improves cow (n=86) and heifer (n=91) embryo production and quality. Time of estrus was synchronized in animals with an ear implant with 3 mg of norgestomet associated with an i.m. injection of 6 mg of norgestomet and 10mg of estradiol valerate (CRESTAR, Intervert International B.V., Boxmeer, Holland) and superovulated by 8 i.m. FSH/LHp injections (400 IU-heifers and 500 IU-cows) in decreasing concentrations at 12h intervals. Animals were inseminated 12 and 24h after observed onset of estrus and embryos recovered 7 days later. Animals were randomly allocated to one of three treatments: (1) vegetable oil vehicle (control), (2) 800 mg of beta-carotene and 500 mg of tocopherol (T800) and (3) 1200 mg of beta-carotene and 750 mg of tocopherol (T1200). Supplemental injections were given at the day norgestomet implants were inserted and at first superovulatory injection. An index (Embryo Quality Index or EQI) was proposed to more precisely evaluate embryo quality (excellent*1 + good*2 + regular*3 + poor*4 + degenerate*5 + unfertilized ova*5)/total. There was an interaction between physiological stage (heifer or cow) and treatment on EQI (P=0.01) and on the proportion of viable embryos (P=0.03), where both variables were improved in T1200 cows, but not in heifers. The average EQI for heifers and cows in control, T800 and T1200 were 2.6+/-0.3 and 3.6+/-0.3; 2.5+/-0.3 and 3.6+/-0.3; 2.9+/-0.3 and 2.7+/-0.3, respectively. The average total number of viable embryos was greater (P=0.01) in supplemented cows (3.5+/-1.1; 5.4+/-1.4 and 7.5+/-1.2 in control, T800 and T1200, respectively), but less (P=0.01) in heifers (7.5+/-1.2; 5.6+/-1.2 and 4.0+/-1.1 in control, T800 and T1200, respectively). Supplementation injections of beta-carotene associated to tocopherol improved embryo quality in superovulated Holstein cows, in the present experimental conditions and may be advantageous in similar embryo production systems. However, at dosages applied in the present experiment, this treatment should not be recommended for nulliparous heifers.

Effect of organic or inorganic trace mineral supplementation on follicular response, ovulation, and embryo production in superovulated Angus heifers.

We determined whether source of trace mineral supplementation prior to embryo collection affected embryo production and quality. Angus half-sibling heifers (n=20) originating from a common herd were assigned to three treatment groups using a 3 x 3 latin square design replicated in time (3 x) and space (6 x complete and 1x incomplete): (1) heifers received no added mineral to their diet (control; n=53); (2) heifers received a commercially available organic mineral supplement (organic; n=52); or (3) heifers received an all inorganic mineral supplement (inorganic; n=55). All heifers had ad libitum access to hay and were fed a supplement containing corn and soybean meal. Treatments were initiated 23 days prior to embryo recovery. Heifers were given a 45-day adaptation period of no mineral supplementation before initiating a new treatment. Ovarian structures were evaluated using transrectal ultrasonography to determine the presence and number of follicles and CL on each ovary. The mean number of recovered ova/embryos was similar among treatments (4.1+/-0.7, 3.8+/-0.7, and 3.3+/-0.7 for control, inorganic, and organic treatments, respectively), the number of unfertilized oocytes was greater (P<0.05) for inorganic (2.3+/-0.5) and control (1.6+/-0.5) treated heifers than organic (0.4+/-0.4) treated heifers. No differences among treatments existed for the number of degenerate or transferable embryos, but individual heifer influenced the total number of embryos/ova, unfertilized ova, and transferable embryos recovered. We conclude that heifer accounted for the greatest differences in embryo production and quality. Source of trace mineral supplementation did not significantly alter embryo number or quality in superovulated purebred Angus heifers fed a well-balanced diet, meeting all trace mineral requirements.

Diets enriched in unsaturated fatty acids enhance early embryonic development in lactating Holstein cows.

We hypothesized that a diet enriched in alpha-linolenic acid would enhance embryonic development relative to diets enriched in linoleic or saturated fatty acids. Twenty-four lactating Holstein cows (86+/-22 d postpartum) were assigned to one of three diets containing saturated fatty acids (SAT; high in palmitic and stearic acids), whole flaxseed (FLX; high in alpha-linolenic acid) or sunflower seed (SUN; high in linoleic acid). Rations were formulated to provide 750 g supplemental fat/cow/d in all dietary groups. Ovulation (Day 0) was synchronized approximately 20 d after diets began. Ultrasound-guided follicular ablation of all follicles >8 mm was performed 5 d after ovulation; super stimulatory treatments began 2 d after follicular ablation, and embryos were collected non-surgically 7 d after AI. Fertilization rate, numbers of follicles and ovulations, and total and transferable embryos did not differ (P>0.05) among dietary groups. Sixty-one transferable embryos were stained and total blastomere number determined. Blastomere number was affected by diet (P<0.01); without regard to stage of development, embryos collected from cows fed SAT had lower (P<0.01) blastomere numbers (mean+/-S.E.M.; 77.1+/-3.9) than those from cows fed FLX (93.4+/-3.3) or SUN (97.2+/-3.5). Differences were most evident in the expanded blastocyst stage; at this stage, embryos of cows fed FLX and SUN diets had more blastomeres (P<0.02) than those of cows fed SAT (115.4+/-6.3, 132.3+/-8.3, and 89.3+/-9.6 cells, respectively). Although our hypothesis was only partially supported, embryonic development was enhanced in Holstein cows fed unsaturated fatty acids compared to those fed saturated fatty acids.

Incidence of severe ovarian hyperstimulation syndrome after GnRH agonist/HMG superovulation for in-vitro fertilization.

In 1673 treatment cycles stimulated with buserelin and HMG, for IVF, GIFT or ZIFT, the severe ovarian hyperstimulation syndrome (OHSS) occurred in 10 cycles (0.6%). Eight patients were hyperandrogenic and showed an increased ovarian response to HMG. After replacement of a maximum of three embryos or zygotes, seven women became pregnant. Three women had a multiple gestation. All patients recovered uneventfully with conservative treatment. Support with progesterone or continuation of the agonist during the luteal phase did not prevent OHSS, confirming that the ovulatory HCG dose is the most important factor in inducing this severe complication. Luteal supplementation with HCG and/or HCG production during implantation could exacerbate OHSS.