Total Embryos Research Articles

145 Comparison of single to multiple injections of follicle-stimulating hormone before ovum pickup in Holstein heifers: Oocyte recovery and embryo production

The demand for invitro-produced embryos from heifers with high genetic merit has increased over time. Synchronization and stimulation of follicular growth before ovum pickup (OPU) has been used to improve oocyte quality and, consequently, embryo production. Multiple injections involve extra labour and stress for both personnel and cattle. The release of FSH can be prolonged by using 0.5% hyaluronan (HA) as a diluent, allowing a decrease in the number of injections. The objective of this study was to compare oocyte recovery and embryo production between single or multiple injections of FSH before OPU of Holstein heifers. During April and May 2020, 20 Holstein heifers (8 to 15 mo old) from Ruann Dairy (Riverdale, CA) were randomly divided and submitted to two different treatments (crossover design). Gonadotrophin-releasing hormone (GnRH; Fertagyl®, Merck, 129µg, IM) was given to synchronize the follicular wave emergence. Treatment 1×FSH consisted of a single intramuscular (IM) injection of 100mg of FSH (Folltropin®, Vetoquinol) 36h after GnRH. The FSH consisted of a 2.5-mL injection of 400mg of FSH diluted in 10mL of 0.5% HA. OPU was performed 48 to 50h after FSH. Treatment 5×FSH consisted of 100mg of FSH divided into 5 equal IM injections (10-14h intervals) 36h after GnRH. The FSH consisted of 5×1-mL injections of 400mg of FSH in 20mL of saline. OPU was performed 18 to 20h after the last FSH injection. All donors received both treatments at a 14-day interval and the recovered oocytes were fertilized with the same sexed female-sorted semen in both rounds. OPU, oocyte classification, IVM, IVF, and culture (IVC) were performed as described by Demetrio et al. (2020 Anim. Reprod. 17, e20200053). All oocytes went into IVM, except for degenerated oocytes. The number of 4-cell (or more) embryos on Day 3 of IVC divided by the number of oocytes in IVC after IVF is defined as the cleavage rate. The number of blastocysts (early to hatched) on Day 7 of IVP divided by the number of oocytes in IVC after IVF is defined as the blastocyst rate. Poisson-normal (count data) and Logistic-normal (proportion data) models were used to analyse the data. Treatment, donor (random effect), and sire were included in the models. The results are summarized in Table 1. There were no differences between the two treatments on the number of oocytes recovered per OPU (total and grade 1 and 2), percentage of grade 1 and 2 oocytes, cleavage rate, blastocyst rate and number of embryos (total and grade 1). Oocyte recovery and embryo production are highly donor dependent. Stimulation of the follicular growth before OPU with one single injection of FSH diluted in 0.5% HA 36h after GnRH can be efficiently used for IVP in Holstein heifers, without decreasing the number of oocytes recovered and/or embryos produced with the advantage of reducing labour and stress of handling cattle. Table 1. Number and quality of oocytes and cleavage and blastocyst rates Treatment OPU Oocytes per donor Grade 1 and 2 oocytes (%) Cleavage rate (%) Blastocyst rate (%) Total embryos per OPU Grade 1 embryos per OPU 1×FSH 20 17.0 45.7 84 39.8 6.2 3.8 5×FSH 20 19.9 46.5 82 35.6 6.3 4.0

146 Single injection of follicle-stimulating hormone before ovum pickup in lactating Holstein donors: Oocyte recovery and embryo production

Lactating donor cows frequently have decreased oocyte quality, lower fertilization rates, and impaired early embryonic development due to their lactational metabolic challenges. Synchronization and stimulation of follicular growth before ovum pickup (OPU) has been used to improve oocyte quality and consequently, embryo production. The objective of this study was to evaluate the effects of a single injection of FSH before OPU on oocyte recovery and embryo production in lactating Holstein donors. During June and July 2020, 22 lactating Holstein donors (open, 40 to 90 DIM, producing >90 lbs of milk) from Ruann Dairy (Riverdale, CA) were randomly assigned to one of two treatments (crossover design). Donors did not receive any injections before OPU when assigned to the No FSH treatment. Treatment 1×FSH consisted of a single intramuscular (IM) injection of 140mg of FSH (Folltropin®, Vetoquinol) 36h after gonadotrophin-releasing hormone (GnRH; Fertagyl, Merck®, 129µg, IM). The FSH consisted of a 3.5-mL IM injection of 400mg of FSH diluted in 10mL of 0.5% hyaluronan (HA). OPU was performed 48 to 52h after FSH. All donors received both treatments on a 14-day interval. The recovered oocytes were fertilized with the same sexed female-sorted semen in both rounds (3 different sires were used). OPU, oocyte classification, IVM, IVF, and culture (IVC) were performed as described by Demetrio et al. (2020 Anim. Reprod. 17, e20200053). All oocytes went into IVM, except for degenerated occytes. The number of 4-cell (or more) embryos on Day 3 of IVC divided by the number of oocytes in IVC after IVF is defined as the cleavage rate. The number of blastocysts (early to hatched) on Day 7 of IVP divided by the number of oocytes in IVC after IVF is defined as the blastocyst rate. Poisson-normal (count data) and Logistic-normal (proportion data) models were used to analyse the data. Treatment, donor (random effect), and sire were included in the models. The results are summarised in Table 1. Oocyte recovery and embryo production were highly donor dependent. There were no differences in the number of recovered oocytes among treatments. Stimulation of the follicular growth before OPU with one single injection of FSH diluted in 0.5% HA 36h after GnRH improved oocyte quality, cleavage rates, blastocyst rates, embryo quality, and the total number of embryos per OPU in lactating Holstein donors. Table 1. Oocyte recovery, cleavage rate, and embryo production results Treatment OPU Oocytes per donor Grade 1 and 2 oocytes Cleavage rate (%) Blastocyst rate (%) Total embryos per OPU Grade 1 embryos per OPU No FSH 22 18.2 30a 69a 22a 3.4a 1.6a 1×FSH 22 17.5 34b 77b 34b 5.3b 3.3b a,bValues with different superscripts in the same column differ (at least P<0.05).

121 Anti-Müllerian hormone cutoff values for the selection of oocyte donors in the Gir (Bos indicus) breed

Anti-Müllerian hormone (AMH) has a well-known association with antral follicle count (AFC) and the number of oocytes recovered by OPU in cattle. However, to be used as a criterion to select candidate donors for ART, AMH reference values must be established for each breed. The aim of this study was to estimate AMH cutoff values for selecting oocyte donors in Gir (Bos indicus) cattle. A blood sample was collected at a random day of oestrous cycle from Gir heifers (n=120) at 23.3±0.5 months of age, and the plasma was stored for further AMH analysis by ELISA, using a commercial kit (AL114, AnshLabs) in a private laboratory (LEAC, Sao Paulo, Brazil). Data from 506 ovum pickup-invitro embryo production (OPU-IVEP) sessions (4.2±0.2 per donor) performed in these heifers from 2017 to 2019, starting 120 days after blood sampling, were then analysed. Donors were ranked in quartiles according to the number of total oocytes recovered, viable oocytes recovered, and embryos produced. Those classified in the first quartile for each endpoint were considered poor responders. The AMH values were then analysed using the receiver operating characteristic (ROC) curve, and cutoff values for each endpoint were estimated based on best values for sensitivity and specificity. A second analysis was performed to check which cutoff value would result in a significant increase in average for each endpoint. The OPU-IVEP data were ranked according to corresponding AMH values (smaller to greater), and the average of the selected group of donors was compared with the original group by ANOVA, using the PROC GLM of SAS (SAS Institute Inc.), after successive exclusion of donors with lower AMH concentrations. Results are shown as mean±s.e.m. On average, heifers presented AMH concentrations of 932.9±48.4pgmL−1 (ranging from 112.7 to 2044.8pgmL−1) and produced 32.3±1.5 total oocytes, 25.9±1.4 viable oocytes, and 5.3±0.4 embryos per OPU-IVEP session. The heifers ranked in the third and last quartiles yielded more total oocytes, viable oocytes, and blastocysts than those ranked in the first quartile (P<0.05). Based on the total number of oocytes recovered (59.2±3.1), heifers of the last quartile had an estimated AFC >60. The area under the ROC curve (AUC) for total oocytes, viable oocytes, and embryos produced were 0.77 (P<0.0001), 0.75 (P<0.0001), and 0.72 (P=0.0003), and the estimated cutoffs for AMH concentration were 761.4, 622.5, and 681.5pgmL−1, respectively. After successive exclusions of donors with lower AMH values, averages were greater (P<0.05) at a cutoff value of 632.4pgmL−1 for total oocytes (36.9±1.6 vs. 32.3±1.5; +15.3%), 672.3pgmL−1 for viable oocytes (30.3±1.4 vs. 25.9±1.4; +19.4%), and 776.8pgmL−1 for embryos produced (6.6±0.3 vs. 5.3±0.4; +23.4%). However, the use of these cutoff values to increase the averages would result in the exclusion of 32.8, 37.9, and 50.0% of the potential donors for each endpoint, respectively. In summary, these results demonstrated that an AMH concentration of approximately 700pgmL−1 can be used to select donors with higher oocyte yield and thus to increase IVEP outcomes in Gir heifers. This research was supported by Fazendas do Basa, Fapemig CVZ APQ 03430-17.

Fixed-time artificial insemination protocols on brazilian locally adapted breed gilts on ovulatory response and embryo production.

The aim of this study was to use estrus synchronization protocols to favor fixed-time artificial insemination and consequently fixed-time embryo collection, and increase embryo production using eCG, in gits. In a cross over design, nine Piau breed gilts were subjected to 18 days of oral progesterone; P4 group did not receive any further; GnRH group received 25µg of GnRH 104 hours after the final application of P4; and eCG+GnRH group received 1000IU of eCG 24 hours after the final P4 in addition to GnRH for subsequent embryo collection, that was performed six days after first AI, by laparotomy. Artificial insemination was performed after 12 and 24 hours of estrus in P4 group, and 128 and 144 hours in GnRH and eCG+GnRH groups. The number of CL (8.6±3.9; 8.3±2.1; 26.7±15.0) and anovulatory follicles (4.3±3.7; 3.9±3.9; 17.2±9.5) was higher in the eCG+GnRH gilts (P<0.05). However, the use of 1000 IU of eCG reduced (P<0.05) the number of total structures (5.2±3.6; 5.1±3.1; 1.7±2.7), viable embryos (5.0±3.5; 4.8±3.3; 0.4±0.7), freezable embryos (3.6±3.4; 3.3±3.8; 0.1±0.3) and recovery rate (63.7±38.9; 58.6±24.7; 5.38±9.5). P4 and GnRH protocols were effective in the production and recovery of embryos. However, the use of 1000 IU of eCG, 24 hours after P4, was not effective in promoting the production of embryos, although the animals had superovulated.

Influence of antral follicle count on in vitro embryo production, sexual precocity and conception rate of Senepol cattle

Context There are contradictory results concerning the relationship between antral follicle count (AFC) and reproductive traits for different breeds of cattle. Understanding this relationship could be useful to selecting animals for breeding. Aims We evaluated the effects of AFC on parameters related to in vitro embryo production (IVP), and the influence of AFC on sexual precocity and conception rate at first fixed timed artificial insemination (FTAI) of Senepol females. Methods Ovarian ultrasound was performed to count antral follicles, and females were classified as low AFC (≤22 follicles), intermediate AFC (23–36 follicles) or high AFC (&gt;36 follicles). Expt 1 evaluated the influence of AFC on IVP, with data from 146 adult cows that were submitted to follicular aspiration. Expt 2 evaluated the influence of AFC on heifer sexual precocity, determined by the presence of a corpus luteum (n = 608), and on conception rate at first FTAI (n = 143). Key results High AFC cows had a greater (P &lt; 0.0001) number of viable oocytes and cleaved embryos and a higher average number of embryos than low AFC cows (38.28 vs 9.92, 38.28 vs 7.75 and 9.34 vs 2.41, respectively). However, the conversion rate (total viable embryos per oocytes) was not affected (P = 0.46) by AFC category. High AFC heifers tended (P = 0.07) to be more sexually precocious than low AFC heifers (16.73% vs 9.47%). An influence of AFC or of AFC category on conception rate at first FTAI was not detected (P = 0.35 and 0.19, respectively). Conclusions Despite greater IVP in high AFC cows, the conversion rate of viable oocytes to IVP was not affected by AFC category. High AFC heifers tended to be more sexually precocious, although AFC did not influence the conception rate at first FTAI. Implications Oocyte quality is probably not influenced by AFC, but selection of animals according to AFC may improve the sexual precocity of the herd.

Embryo deformities and nesting trends in Kemp’s ridley sea turtles Lepidochelys kempii before and after the Deepwater Horizon oil spill

Kemp’s ridley sea turtlesLepidochelys kempiiwere disproportionately affected by theDeepwater Horizon(DWH) oil spill, which began on 20 April 2010. Embryo deformities were documented in inviableL. kempiieggs before (2008-2010) and after (2011-2013) the DWH spill in 2 Texas (USA) nesting areas (Upper Texas Coast and Padre Island National Seashore). Additional nesting trends, including clutch size and hatching success, were also investigated. Total and late-stage embryo deformity prevalence were 1.5 times greater after 2010 than before, but low in all nesting seasons (mean ± SD: 0.7 ± 8.5% total; 0.6 ± 8.0% late-stage) and did not differ between locations. Craniofacial and carapace deformities were the most frequently observed deformity types. Documented nests in both areas declined in 2010 relative to previous years, ending an exponential increase observed beginning in 1995. Clutch size remained consistent before and after the spill. Hatching success averaged 87.0 ± 33.3% in all years, but no effects from DWH were determined. Collectively, these data represent useful benchmarks against which to judge impacts of future crude oil spills and other catastrophic events.

Effect of Cryopreservation on Olive (Olea europaea L.) Plant Regeneration via Somatic Embryogenesis.

Olive somatic embryos have been successfully cryopreserved using the droplet-vitrification method on aluminum foil strips. Although acceptable recovery rates have been obtained after rewarming, the influence of this cryopreservation protocol on the somatic embryogenesis process is unknown. To evaluate the effect of cryopreservation on olive somatic embryogenesis, the behavior of cultures established from cryopreserved somatic embryos was compared with that of control, non-cryopreserved cultures in the different phases of the somatic embryogenesis process. In order to analyze the influence of the genotype, this investigation was carried out in two independent lines. During the proliferation step, only the line T1 was affected by cryopreservation, with higher fresh weight increases. Although similar total embryos were produced per culture, freezing in liquid nitrogen significantly improved the maturation pattern in the line P5. Better germination results were also found in this embryogenic line. The genotype plays a key role, largely determining the effect of cryopreservation on olive somatic embryogenesis. A specific genotype-dependent response was found depending on the culture step. Variations observed could not be associated to differences in the embryogenic lines’ instability to maintain their morphogenic competence after cryopreservation. Embryogenic cultures established after rewarming retained their regeneration capacity, with no evident negative effects affecting their regeneration capacity.

Effects of semen dosage, oviductal sperm storage and insemination interval on egg fertility, embryo mortality and hatchability in Nera Black breeder chickens

The biological basis of sustaining fertility in poultry is their ability to store sperm cells in the sperm storage tubules (SST) located in the uterovaginal junction. However, artificial insemination in poultry industry is haphazardly administered in Nigeria without regulation on semen dose and frequency of insemination for optimum fertility. The objective of this study was to establish a semen dosage and insemination interval for maximum fertility and embryonic survival in Nera black layer breeder chickens. A total of 80 breeder hens (52 weeks) were allotted to five (5) treatments with four (4) replicate per treatment. Semen was pooled from 10 matured breeder cocks and inseminated to four groups of hens at varied semen dose of 0.02mL (T1), 0.04mL (T2), 0.06mL (T3) and 0.08mL (T4) of undiluted semen while hens in T5 were mated naturally, both for two consecutive days. 0.02, 0.04, 0.06 and 0.08mL of pooled semen contained 20.43×10 , 40.87×106 , 61.30×106 and 81.74×106 motile spermatozoa. Eggs were collected, stored and artificially incubated weekly for 4 weeks. Fertility, embryo mortality and hatchability parameters were determined. Another 78 breeder hens were allocated into 4 treatments of 5 replicates per treatment with unequal number of hens and were inseminated with 0.02mL of raw semen containing 20.43 × 106 motile sperm cells at 3, 6, 9 and 12 days intervals. Fertility, hatchability and embryo mortality were determined. Data were analysed using descriptive statistics and ANOVA at &amp;0.05 Hatch of fertile eggs in T5 at week 2 (65.36±13.28) was significantly higher (p&lt;0.05) . than T1 (33.83±12.65), T2 (13.25±6.88), T3 (39.17±14.17) and T4 (28.21±11.37). At weeks 1 and 2, there was no significant different across the treatments. Fertility at 4 weeks in T1 (11.53±6.66) was significantly (p&lt;0.05) different from treatments T2 (0.00±0.00), T3 (0.00±0.00), T4 (1.66±1.66) and T5 (0.00±0.00). Total and early embryo mortality in week 3 was significantly higher (p&lt;0.05) in T1 (100.00±0.00, 95.00±5.00) than in treatments T2 (43.75±0.00, 43.75±25.77), T3 (66.67±23.57, 66.67±23.57), T4 (95.00±5.00, 85.00±15.00) and T5 (37.50±23.94, 22.92±15.73). Fertility was significantly (p&lt;0.05) higher in 3 days insemination interval (52.65±7.25) compared with 6 days (39.87±4.70), 9 days (22.98±5.71) and 12 days (36.14±6.89). At weeks 1 and 3, the hatch of fertile eggs across the treatments was not significantly (p&gt;0.05) different from one another. This study suggests that 6 inseminating semen dose of 0.02mL containing approximately 20.43×10 motile sperm cells in Nera black layer breeder chickens will give a maximum fertile period of 5 days, while insemination interval of 3 days using 0.02mLof semen gave highest fertility level.

Transcript abundance of anti-Mullérian hormone and follicle-stimulating hormone receptor predicted superstimulatory response in embryo donor Holstein cows.

The goal was to investigate the relationship among mRNA expressions of anti-Mullérian hormone (AMH), follicle-stimulating hormone receptor (FSHR) and responses to superovulation (SO) in embryo donor dairy cows. Holstein cows (n=19) were submitted to a standard SO protocol, with twice daily FSH treatments, and artificially inseminated. Prior to SO (Day 0), relative mRNA expressions of AMH and FSHR in blood were determined for all cows. Day 7 embryos were collected and were graded to determine superovulatory response for each donor. Results showed that relative mRNA expressions of AMH and FSHR were positively correlated (R2 =0.94). Relative mRNA expressions of both AMH and FSHR were positively correlated with total embryos (R2 =0.68 and 0.69, respectively), total transferable embryos (R2 =0.92 and 0.97, respectively) and total grade 1 embryos (R2 =0.54 and 0.59, respectively). Further, transcript abundances of AMH and FSHR positively associated with milk production of donor cows, and meanwhile, they were negatively associated with days in milk (DIM) at submission of cows to SO (p<.05) protocol. The relative mRNA expression of AMH was higher (p<.05) in donor cows<5 years of age. However, age of donor at superovulation did not influence mRNA expression of FSHR. Collectively, we infer that the mRNA expressions of AMH and FSHR prior to superovulation can predict donor cows' positive response to superovulation.

The Response of Donor Holstein Cows to Ovarian Super-Stimulation Using Two Different Protocols GnRH and Estradiol

The objective of the present study was planned to compare the response of donor Holstein cows to a GnRH-based (GnRH-SOV, n=9) and estradiol-based (E2-SOV, n=10) protocols. The super-stimulation protocols consisted of twice daily decreasing doses of porcine FSH with a total dose of 400 mg and the cows in both groups were sampled three times, before initiation of superovulation, at estrus and at embryo collection from donors. Circulating levels of FSH, LH, progesterone, estrogen and anti-Mullerian hormone (AMH), as well as response to superovulation were compared between the two groups. Results revealed that embryo yield and quality did not differ (P<0.05) between the two groups. When circulating concentrations of hormonal biomarkers were tested for possible correlations with embryo quality or quantity, progesterone was correlated with total embryo (r=0.71, P<0.01), total transferable embryo (r=0.84, P<0.01), second grade and third grade embryo (r=0.72 and 0.73, respectively; P<0.01) regardless of the time of sampling. Strong positive correlations were recorded between circulating levels of AMH prior to superovulation and embryo yield (total embryo, r=0.50; P<0.01), and quality (first grade embryo, r=0.78, P<0.01; second grade and third grade embryo, r=0.47 and 0.50, respectively with P<0.05). In conclusion, donor Holstein cows responded successfully to GnRH-SOV and E2-SOV protocols and circulating levels of progesterone and AMH could predict donor response to superovulation in Holstein cows.

Short communication: Does previous superovulation affect fertility in dairy heifers?

The aim of this study was to evaluate the potential negative effects of superovulation on subsequent fertility of dairy heifers. Holstein heifers (n = 1,783), 312 to 387 d of age, and 273 to 307 kg of body weight (BW) from 2 commercial farms were enrolled. These animals were first selected to be donors (446) or nondonors (CON, n = 1,327) according to their genomic values. Nondonor heifers (CON) were artificially inseminated (AI) according to standard procedures of each farm after reaching 320 kg of BW. Donor heifers were superovulated using a fixed FSH dose (180 mg NIH-FSH-P1; Folltropin-V, Vetoquinol Brasil, Mairiporã, SP, Brazil) and embryos were collected following standard procedures. Heifers that produced fewer than 8 viable embryos after first superovulation (SOV1, n = 337) were no longer used as donors, whereas the remaining heifers (SOV2, n = 109) were superovulated a second time within an interval of 48 to 54 d. Donor heifers (SOV1 and SOV2) were AI once they reached 320 kg of BW, at least 15 d after the last embryo collection. Data on age at first AI, at conception, and at parturition, as well as the number of services per conception, were analyzed by ANOVA, using the PROC MIXED procedure of SAS (SAS Institute Inc., Cary, NC) procedure. Binomial variables (pregnancy per AI, overall pregnancy rate, open heifers at 500 d age, and late pregnancy loss) were analyzed using the GLIMMIX procedure of SAS. The heifers selected to undergo superovulation twice (SOV2) yielded more total (12.6 ± 5.3 vs. 6.8 ± 4.6; respectively) and viable embryos (8.5 ± 3.8 vs. 3.9 ± 2.8; respectively) than those superovulated only once (SOV1). Age at first AI, conception, and at parturition was greater in SOV2, but not in SOV1 compared with nondonor controls. In addition, pregnancy per first AI, overall pregnancy rate, services per conception, open heifers at 500 d of age, and occurrence of pregnancy losses after 60 d of gestation were similar among CON, SOV1, and SOV2 heifers. In summary, a single superovulation performed before heifers reach a minimum weight for breeding did not affect age at conception, calving or other indicators of reproductive efficiency. On the other hand, heifers superovulated twice were first inseminated at a later age than their birth cohorts, but had similar reproductive performance.

Symposium 3: Contexts, Consequences, and Correlates: Examining multiple levels of analysis to understand stress and HPA patterns across development

The objective was to determine associations between response to superovulation and body condition, subclinical endometritis and circulating metabolic biomarkers [adiponectin, leptin, insulin, IGF1, tumor necrosis factor (TNF) α, interleukin (IL) 1β, IL6, and urea] in lactating dairy cows. Ten multiparous lactating Holstein cows in each body condition score (1–5; 1 emaciated; 5 obese) category (BCSC) 2.00 to < 2.50 (BCSC1), 2.50 to < 3.00 (BCSC2), 3.00 to <3.50 (BCSC3), 3.50 to <4.00 (BCSC4) and 4.00 to 5.00 (BCSC5) groups (total n = 50) were randomly selected and superovulated, timed artificially inseminated with frozen-thawed semen from three sires and embryos collected (n = 50 collections). At embryo collection, blood samples and embryo recovery fluid were collected for determination of metabolic markers and presence of subclinical endometritis (lavage technique; > 6% PMN). In total, 379 embryos were collected (average of 7.6 embryos per superovulation). Mean numbers of total ova and embryos was greater for cows in BCSC2, BCSC3 and BCSC4 groups compared with cows in BCSC1 and BCSC5 groups (P < 0.01). Total number of transferrable embryos were greater for cows in BCSC 2 and BCSC3 groups compared with cows in BCSC1, BCSC4 and BCSC5 groups (P < 0.01). Mean number of total ova and embryos and of transferrable embryos was higher for cows with 0 or 1–6% PMN compared to cows with >6% PMN (P < 0.01). In addition, there was a quadratic association between blood urea nitrogen concentrations and % transferrable embryos (r2 = 0.85; P < 0.05) and between BCS and % transferrable embryos (r2 = 0.73; P < 0.05). Circulating adiponectin, leptin, insulin, IGF1 and TNFα were greater in cows with moderate to good body condition compared to thin or obese cows (P < 0.05). Circulating adiponectin, leptin, IGF1 and insulin were greater in normal cows (≤6% PMNs), whereas, TNFα and IL1β and IL6 were greater in cows with subclinical endometritis (P < 0.05). In conclusion, BCS and subclinical endometrial inflammation were associated with superovulatory response and embryo quality. Further, circulating metabolic biomarkers were associated with superovulatory response and embryo quality, likely due to donor’s metabolic status and uterine environment. Optimizing superovulatory responses and embryo quality in lactating dairy cows requires management of nutrition and uterine health.

Intra cytoplasmic sperm injection (ICSI) outcome comparison among normal, poor and hyper responder females following controlled ovarian stimulation using GnRH antagonist protocol

The aim of any given ICSI cycle is to achieve normal response and avoid poor or hyper response. A part from ovarian hyper stimulation syndrome (OHSS), pregnancy rate in hyper responder females usually low which could be related to the adverse effect of hyper estrogenic state on oocytes, embryos quality and uterine endometrium. To evaluate the effect of hyper response on ICSI outcome represented by oocytes, embryos quality, fertilization and pregnancy rates. Study showed that total number of retrieved oocytes, mature, immature oocytes, total embryos, good quality embryos and blastocyst count were significantly higher in hyper followed by normal and poor responders respectively. Fertilization rate was significantly higher in both hyper and normal responders than poor ones, while pregnancy rate was significantly higher in hyper responders 47.6% followed by normal 40.7% and then poor ones 16.7%. Conclusions: Irrespective of abnormal hormonal milieu, excessive ovarian response and higher number of produced oocytes improve ICSI outcome in hyper responder females by increasing the chance of getting higher number of embryos, good quality embryos and even blastocyst numbers which in turn increase the chance of pregnancy

The Corelation Between Serum Estradiol (C23H32O3) Impact on Selected Biomarkers and Outcome of Fresh Embryo Transfer in Comparison with Freeze All Strategy in in vitro Fertilisation Patients

In stimulated cycles the endometrium could be more advanced than the embryos, with a possible negative impact on their implantation capacity. Therefore, there is an ongoing debate regarding the best transfer strategy: freeze-all versus fresh embryos transfer. Our study aimed to analyse if the frozen only embryos-transfer strategy for in vitro fertilisation (IVF) has higher clinical pregnancy rate (CPR) than the traditionally fresh transfer. We performed a retrospective study in a private centre of reproductive medicine. We included only patients who performed fresh embryo transfers (n=245) and patients with all the embryos frozen and then transferred into a non-stimulated cycle (n=117). The reasons for delayed transfer were an increased risk of ovarian hyperstimulation syndrome or the increase of late follicular phase serum progesterone level. The mean age of the study group was 34.33�b4.28 years and mean AMH was 3.68�b3.56 ng/mL. Patients with fresh transfer were significantly older (p[0.0001) and obtained significantly lower number of oocytes at egg retrieval (p[0.0001) and lower zygotes number (p[0.0001) in comparison with patients with frozen transfer. After adjustment for confounders, CPR was significantly higher in patients with fresh transfer in comparison with those with frozen transfer in total group (OR 2.7, p=0.001) and in patients with cleavage stage embryo transfer (OR 6, p=0.008), but not in patients with blastocyst transfer. Similarly, the implantation rate was significantly higher in total group and in both subgroups (p[0.0001). Our study shows that the freeze all strategy should not be performed in all the patients, being inferior to fresh transfer in the total study group and cleavage stage embryo transfers. The decision to transfer frozen embryos should take into account the availability of blastocysts for transfer and the risk benefit profile.

EFFECT OF ADDING THYMUS VULGARIS POWDER TO RATION ON REPRODUCTIVE PERFORMANCE OF QUAIL

This study was conducted in the Poultry farm of Animal production Dept. College of Agriculture and Forestry, Mosul University in order to investigate the effect of adding Thymus vulgaris Powder to the diets on productive performance and Reproductive for laying quail. One hundred forty four unsexed quail Coturnix coturnix 90 day old were randomly distributed into four treatments with four replicate in each replicate nine birds 6 female and 3 male, The treatments were as follows: first basal diet without addition as (control). Second, third and fourth treatments basal diet and addition of Thymus vulgaris Powder in 0.5, 1, 1.5 % respectively. Feed and water was ad libitum. Statistical analysis of data showed significant improvement (p≤0.05) in total egg number, average egg weight, hen day production, feed conversion ratio(g feed: g eggs) for treatments third and forth as compared with control. Also significant increase in daily and total egg mass in treatments second, third and forth as compared with control. No significant difference in live body weight, average body weight gain, feed consumption , feed conversion ratio (g feed: egg), mortality rate, fertility percentage, hatchability percentage from total and fertile eggs and embryo mortality

Single Embryo Transfer Versus Double Embryo Transfer: A Cost-Effectiveness Analysis in a Non-IVF Insurance Mandated System.

Because of increased morbidity seen in multiple gestations, the American Society of Reproductive Medicine recommends transfer of blastocysts one at a time for most patients. While cost-effectiveness models have compared single embryo transfer (SET) versus double embryo transfer (DET), few incorporate maternal and neonatal morbidity, and none have been performed in U.S. Military facilities. The purpose of this study was to determine the cost effectiveness of sequential SET versus DET in a U.S. Military treatment facility. A cost-effectiveness model was created based on 250 patients between the ages of 20-44 who previously underwent in vitro fertilization (IVF) at our facility. The model consisted of patients pursuing either SET or DET with two total embryos. Cycle outcomes were determined using the published SARTCORS success calculator. Neonatal and obstetrical outcomes were simulated based on singleton and twin IVF pregnancies. Neonatal and obstetrical cost estimates were based on internal data as well. If 250 model patients pursue SET, 140 live births would occur, with total cost of $5.7 million, and cost per delivery of $40,500. If the model patients pursued DET, 117 live births would occur, with total cost of $9.2 million and a cost per delivery of $77.700. DET would lead to more total infants (207 vs. 143 in SET cohort). Personal costs are higher in SET versus DET cohorts ($23,036 vs. $20,535). SET in a system with no infertility coverage saves approximately $3.5 million per 250 patients. Higher personal costs as seen with SET may incentivize patients to seek DET. The total savings should encourage alteration to practice patterns with the U.S Military Healthcare System.

Effect of equine chorionic gonadotropin on glycosidase activity in the reproductive tract of ewes, in relation to ovarian response and embryo yield

Abstract The aim of this study was to investigate a) the effect of equine Chorionic Gonadotropin (eCG), in the dosage used for oestrus synchronization or for superovulation, on the activity of α-mannosidase (α-MAN) and β-N-acetyloglucosaminidase (β-NAGASE) in the genital tract of ewes before maternal recognition of pregnancy and b) the possible relationships among ovarian steroids concentration, glycosidase activity and embryo yield and quality. Twenty-four Chios ewes were treated with progestagen intravaginal sponges for 12 days and eCG [superovulation (SOV), n = 7, 1000 IU; oestrus synchronization (OS), n = 7, 500 IU; controls (C), n = 10, 0 IU] at sponge removal. Ovarian response was assessed and embryos were collected on day 6 of the oestrous cycle, after slaughter. Oestradiol-17β and progesterone concentrations were determined in blood serum samples collected before slaughter using a radioimmunoassay. The activities of α-MAN and β-NAGASE were assayed, photometrically, in uterine horn flushing (UHF) samples, caruncular endometrium (CE) and intercaruncular (ICE) endometrium samples, collected after slaughter. Alpha-MAN activity was higher in the UHF, CE and ICE of SOV ewes, as well as in the UHF and ICE of OS ewes, compared to controls. Beta-NAGASE activity in the UHF and in the ICE of SOV ewes was lower than in C ewes. Alpha-MAN activity in the UHF was positively correlated with the response to superovulation in terms of total recovered structures, embryos or high-quality embryos collected from the ewes. Beta-NAGASE activity in the CE correlated to the number of corpora lutea observed on the ovaries, at the day of embryo collection. Beta-NAGASE activity in UHF and α-MAN activity in the ICE were negatively related to oestradiol-17β concentration in blood serum; β-NAGASE activity in UHF and in ICE was negatively related to progesterone concentration in blood serum. In conclusion, eCG in the dosage used for superovulation and, in a lesser degree, in the dosage used for oestrus synchronization affects glycosidase activity in the genital tract of Chios breed ewes, before maternal recognition of pregnancy; glycosidase activity in the genital tract correlate to ovarian response and embryo yield. Further research is necessary to fully clarify the interrelationship between ovarian steroids concentration and glycosidase activity in the genital tract of ewes after hormonal treatment.

The addition of clomiphene citrate to ovarian stimulation protocols for poor responders

Poor ovarian response (POR) is one of the most challenging problems in assisted reproduction. Several strategies have been used to improve pregnancy rates. The use of Clomiphene Citrate (CC) has been shown to improve ovarian stimulation outcomes and decrease gonadotropin requirements in women of advanced reproductive age. However, the combination of CC and gonadotropins to improve pregnancy rates after in IVF in poor responders is still unexplored due to the small number of trials with few participants.This is a prospective cohort trial involving 12 patients diagnosed with poor ovarian response who underwent ovarian stimulation during the period between June 2015 and September of 2017. All patients were treated with the maximum dose of gonadotropins (hMG, 300 IU/day, hMG group) according to a short gonadotropin/GnRH antagonist protocol. In a subsequent cycle those patients underwent the same stimulation protocol with the addition of 100 mg of CC from day 3 to day 7 (CC-hMG group).Supplementation with 100 mg of CC resulted in a statistically significant increase in estradiol levels, number of follicles and number of oocytes retrieved, as well as an increase in the number of total embryos available for transfer. Furthermore, a significant reduction was observed in cancellation rates in the CC-hMG group. Two clinical pregnancies, which resulted in two live births and 3 biochemical pregnancies were achieved in the CC/hMG group.Furthermore, by employing open-source, biological data we identified a common gene (Estrogen Receptor 1, ESR1) between genetic targets of clomiphene treatment and POR which could explain the benefits of clomiphene in this group of patients.In conclusion, the addition of CC 100 mg to the stimulation regimen in women diagnosed with POR and previous failed IVF cycles could improve stimulation results, but this study could not demonstrate any benefit in terms of clinical pregnancies and live births. The effectiveness of this treatment requires further investigation.

Effects of flush feeding strategy before breeding on reproductive performance of modern replacement gilts: impacts on ovulation rate and litter traits

AbstractThe effects of two feed levels offered during two estrous cycles before insemination were evaluated on the reproductive performance of gilts. A total of 93 gilts (PIC Hendersonville, TN) were individually housed and manually fed twice a day with 2.1 or 3.6 kg/d of a corn and soybean meal-based diet (3.15 Mcal ME/kg and 0.64% standardized ileal digestible lysine), during two estrous cycles before breeding (cycle 1, between first and second estrus; cycle 2, between second and third estrus). Gilts were weighed at the beginning of the experiment, at second and third estrus, and at slaughter (30.2 ± 1.2 d of gestation). Follicles were counted at second estrus, and the embryo-placental units and the corpora lutea were individually counted, measured, and weighed at slaughter. Gilts fed 3.6 kg/d had greater BW gain during cycle 1 and cycle 2 (P < 0.001; + 9.8 kg and + 10.0 kg, respectively) becoming heavier at second and third estrus (P < 0.001). At second estrus, gilts fed 3.6 kg/d had 1.6 more medium-large follicles (P = 0.074) but no difference in follicle size (P = 0.530) was observed. Gilts fed 3.6 kg/d in cycle 1 or cycle 2 had a greater ovulation rate at third estrus (P < 0.016) than those receiving 2.1 kg/d. Also, 3.6 kg/d in cycle 2 increased early embryo mortality (P = 0.006; 2.3 vs. 1.1 dead embryos) and consequently reduced total embryo survival (P = 0.002; 84.6 vs. 90.1%). Gilts fed 3.6 kg/d during cycle 1 had two more total embryos (P < 0.001; 17.2 vs. 15.1) and two more vital embryos on day 30 (P < 0.001; 16.7 vs. 14.5) in comparison with gilts fed 2.1 kg/d. The coefficient of variation for placental length was greater for gilts fed 3.6 kg/d during cycle 1 (P = 0.003). No further significant effects of feeding levels were observed on embryo and placental traits (P ≥ 0.063). These results suggest that the feeding level during the first cycle after pubertal estrus is crucial to set ovulation rate and potential litter size for breeding at next estrus. However, flush feeding gilts before insemination can negatively impact litter size by reducing embryo survival when breeding at third estrus.

Intraovarian influence of bovine corpus luteum on oocyte morphometry and developmental competence, embryo production and cryotolerance

Three experiments were conducted to determine influence of the bovine corpus luteum (CL) on morphometric and functional characteristics of oocytes, and subsequent embryonic development. Cumulus-oocyte complexes were aspirated from two types of cows: 1) with a CL in one ovary (CL+) and without a CL in the contralateral ovary (CL-), 2) and from cows without CL in either ovary (C). Intracellular activity of the enzyme glucose-6-phosphate dehydrogenase (G6PDH), oocyte diameter and thickness of the zona pellucida were determined (Experiment 1). Then, the rate of in vitro oocyte maturation for each ovarian category was evaluated and oocyte diameter and zona pellucida thickness were measured after maturation (Experiment 2). In Experiment 3, in vitro embryo production and cryotolerance were assessed. The oocyte diameter was greater (P < 0.01) and the zona pellucida was thinner in CL+ than in CL- (P > 0.05) or C (P = 0.0131) ovaries. Activity of G6PDH was lower in oocytes from CL+ than CL- (P < 0.01) and C (P = 0.0148) ovaries. Rate of oocyte maturation, oocyte diameter and thickness of the zona pellucida after maturation did not differ among groups. Rate of cleavage was greater in zygotes from CL+ than from CL- or C (P < 0.01); and CL+ ovaries produced more total embryos on day 7 (P < 0.05) and more blastocysts (P < 0.01) than CL- and C ovaries. Rate of expansion and hatching of day-7 vitrified-warmed blastocysts at 24 and 48 h of culture did not differ among groups. In conclusion, oocytes collected from CL+ ovaries were larger and metabolically more prepared to continue maturation than those from ovaries lacking a CL. Also, rates of cleavage and yield of blastocysts were greater for oocytes from CL+ ovaries than from CL- and C ovaries. These findings indicate that a CL influenced oocyte developmental competence and embryonic development, presumably through intraovarian interactions.