To evaluate In Vitro Fertilization (IVF) outcomes stratified by age in women who integrated Whole Systems Traditional Chinese Medicine (WSTCM).DESIGN: Secondary analysis of a retrospective patient cohort from 2005 to 2010.MATERIALS AND METHODS: A multi-modal intervention, WSTCM is individualized acupuncture combined with diet, lifestyle, nutritional supplements, and Chinese herbs, if appropriate.We reviewed 1,069 IVF patient charts to assess the effect of adjuvant WSTCM (N¼119) on IVF live births compared with IVF alone (N¼580) or day of ET acupuncture (N¼370) and stratified by age group (<35, 35-37, 38-40, 41-42, >43 years old).Donor egg (n¼225), frozen (N¼251), cycles with no transfer (n¼312) were excluded.The primary outcome was live birth.Groups were compared using Fisher exact and Chi-square, p<0.05 was significant.Treatment effect was assessed using multivariate logistic regression, adjusted by covariates associated with outcome.Nonsignificant covariates were removed from the model using a stepwise method.RESULTS: Demographics and characteristics were comparable on all variables except: total antral follicles (p¼0.0497),diminished ovarian reserve diagnosis (p¼0.01),other diagnosis (p¼0.03),prior IVF (p¼0.004),number of mature eggs (p¼0.02), and number of embryos transferred (p¼0.003).IVF augmented with WSTCM was associated with an increase in live births in 38to 40-year-old women compared with IVF alone (p¼0.04), and in 41-to 42year-old women compared to all cycles (p¼0.01).There were trends toward a significant difference in live births in the 38-40 age group compared with all cycles (p¼0.05), and clinically relevant differences in the <35 and 35-37 age groups with WSTCM compared with the other groups, though not significant.Confidence intervals were broad, therefore larger populations are needed to confirm these observations.CONCLUSIONS: Age may be a consideration when integrating acupuncture or WSTCM with IVF and may benefit women aged 38 and older.In this retrospective study, adjuvant WSTCM and IVF was associated with increased odds of live birth in women aged 38 to 42 years old.Small group numbers limit the strength of these observations.

This study describes the localization of the U2 small nuclear RNA (snRNA) and the major U snRNA group ribonucleoproteins (snRNPs) during bovine preimplantation development. In vitro maturation, fertilization, and oviductal epithelial cell coculture methods were employed to produce several developmental series totalling over 2,000 preimplantation‐stage bovine oocytes and embryos. These oocytes and preimplantation embryos were processed for in situ hybridization, immunofluorescence and Northern blotting methods. The U2 snRNA and the major U group snRNPS were localized initially over the germinal vesicle (GV) of preovulatory oocytes but following GV breakdown were released throughout the ooplasm. They subsequently reassociated with both pronuclei during fertilization. From the two‐cell to the blastocyst stages, the U2 snRNA and U snRNPs were localized to the interphase nucleus of each blastomere. The levels of U2 snRNA throughout bovine preimplantation development were determined by probing a Northern blot containing total RNA isolated from the following preimplantation bovine embryo stages: one to two cell, eight to 16 cell, early morula (>32 cell), and late morula/early blastocysts. The levels of U2 snRNA remained constant between the one‐cell and eight‐to 16‐cell bovine embryo stages but increased 4.4‐fold between the eight‐ to 16‐cell stage and the late morula/early blastocyst stages. The results suggest that a maternal pool of snRNAs is maintained in mammalian preimplantation embryos regardless of the duration of maternal control of development.

The sensitive technique of mRNA phenotyping with the reverse transcription-polymerase chain reaction was employed to determine the patterns of gene expression for several growth factor ligand and receptor genes during bovine preimplantation development. Several thousand bovine embryos encompassing a developmental series from one-cell zygotes to hatched blastocysts were produced by the application of in vitro maturation, fertilization, and oviductal epithelial cell embryo coculture methods. Transcripts for transforming growth factor (TGF-alpha) and platelet-derived growth factor (PDGF-A) are detectable in all preimplantation bovine stages as observed in the mouse. Transcripts for TGF-beta-2 and insulin-like growth factor (IGF-II) and the receptors for PDGF-alpha, insulin, IGF-I, and IGF-II are also detectable throughout bovine preimplantation development, suggesting that these mRNAs are products of both the maternal and the embryonic genomes in the cow, whereas in the mouse they are present only following the activation of the embryonic genome at the two-cell stage. In contrast to the mouse embryo, IGF-I mRNA was detected within preimplantation bovine embryos. Basic fibroblast growth factor (bFGF) is a maternal message in the bovine embryo, since it is only detectable up until the eight-cell embryo stage. Bovine trophoblast protein (bTP) mRNA was detectable within day 8 bovine blastocysts. As was observed in the mouse, the transcripts for insulin, epidermal growth factor (EGF), or nerve growth factor (NGF) were not detectable in any bovine embryo stage. Analyses of this type should aid the development of a completely defined culture medium for the more efficient production of preimplantation bovine embryos.

A total of 901 cumulus-oocyte complexes (COCs) were collected from bovine ovaries obtained at a local abattoir. COCs randomly assigned to Treatment I (n = 451), were cultured in TCM-199 + 10% fetal bovine serum (FBS) and hormones, while oocytes in Treatment II (n = 450) were cultured in TCM-199 + 20% estrous cow serum (ECS). Assessment of maturation revealed that 91.3% (42/46) of oocytes in Treatment I had reached metaphase II of meiosis, which was greater (P < 0.05) than the 73.3% (33/45) in Treatment II. Following in vitro fertilization, 203 oocytes from Treatment I were co-cultured on bovine granulosa cells (Treatment IA) while the remaining 202 oocytes were co-cultured on bovine oviductal cells (Treatment IB). Similarly, 203 oocytes from Treatment II were co-cultured on granulosa cells (Treatment IIA) or oviductal cells (Treatment IIB, n = 202). Co-culture was maintained for 8 days. The proportion of cleaved zygotes was higher (P < 0.05) in Treatment IB (86.6%) compared to Treatments IA (78.8%), IIA (58.1%), and IIB (64.8%). The proportion of cleaved zygotes that progressed beyond the 16-cell stage was also greater (P < 0.001) in Treatment IB (71.4%) compared to Treatments IA (50.0%), IIA (35.4%) and IIB (55.8%). Treatment IB also produced the highest proportion of blastocysts (P < 0.0001) (41.1%) versus 24.6% (IA), 11.3% (IIA) and 18.3% (IIB). The proportion of day 6 morulae that progressed to form day 8 blastocysts was similar for both co-culture treatments (IA, 70.1%; IB 70.2%; IIA, 51.5%; IIB 50.8%) and varied only between in vitro maturation groups. Results indicate that the combination of in vitro maturation in TCM-99 + 10% FBS and hormones followed by co-culture on oviductal monolayers is a superior system for the production of early bovine embryos.

This study investigated the embryotropic potential(s) of egg yolk (EY) or its fractions, yolk plasma (YP) and yolk granules (YG), in culturing bovine embryos in vitro and substituting for protein (FCS, BSA, and BME-E and MEM-NE amino acids) and energy (glutamine, pyruvate and l-lactate) supplements commonly added to culture medium. In the first set of experiments (Experiment 1, 2 and 3) CR1aa with buffalo rat liver (BRL) cells were used as a co-culture system. The addition of 2.5% or 5% EY significantly increased (P<0.05) blastocyst percent over the BRL control (48.3% and 52.4% vs. 32.5%, respectively). The addition of 5% EY in the absence of FCS and BSA resulted in percent development to blastocysts and hatched blastocysts similar (P>0.05) to those of the BRL control (37.6% and 57.4% vs. 51.5%, 22.7% and 39.5%, respectively). The supplementation of the BRL control with 5% YP compared to that of EY resulted in comparable (P>0.05) percentages of blastocysts and hatched blastocysts (39.0% and 51.6% vs. 40.0% and 58.3%, respectively). In the second set of experiments, the embryotropic potential of YP was examined using a cell-free culture system and a simple salt solution (SS) of NaCl, KCl and NaHCO3 as the base medium. The supplementation of an energy-supplemented cell-free simple salt solution (E-SS) with 5% YP in the absence of supplemental protein resulted in percent development into blastocysts and hatched blastocysts comparable (P>0.05) to those of a BRL control (39.2% and 15.8% vs. 37.1% and 22.2%, respectively). The addition of YP to the simple salt solution with hemicalcium l-lactate as the only supplemented energy ingredient resulted in percentages of blastocysts and hatched blastocysts similar (P>0.05) to those obtained by the supplementation of all energy sources (27.4% and 15.6% vs. 36.4% and 14.0%, respectively). Increasing hemicalcium l-lactate level from 5 to 10, 20 or 25 mM resulted in a significant decrease (P<0.05) in percent development into blastocysts (36.5% vs. 24.8%, 11.6% and 6.7%, respectively). In conclusion, YP, with the advantage of being clearer than EY, is capable of sustaining embryo development to the blastocyst stage in a simple salt solution of NaCl, KCl and NaHCO3 supplemented with hemicalcium l-lactate.

Monosaccharide composition analysis is indispensable for the structural characterization and bioactive studies of polysaccharides. Besides, it is also helpful for the quality control of functional polysaccharides. The process is usually carried out by hydrolyzing the polysaccharides into monosaccharides, and then detecting liberated monosaccharides or their derivatives. However, the accuracy is not guaranteed owing to improper experimental methods that result in the incomplete cleavage of glycosidic linkages, the instability of released monosaccharides and the deviation of detection. A main challenge of monosaccharide composition analysis is the lack of generally applicable hydrolysis and detection methods. Therefore, practical suggestions on how to determine monosaccharide composition of polysaccharides should be proposed. In this review, the hydrolysis conditions and detection methods, along with their usability and limitation, are thoroughly compared and summarized. Finally, future perspectives and remaining challenges in this field were outlined. This review will provide guidelines for monosaccharide composition analysis of polysaccharides from natural sources.

Assisted reproductive technologies have played a major role in the genetic improvement of livestock as well as in the development of transgenic animal technologies. Artificial insemination (AI) was the first widely-accepted assisted reproductive technology for livestock, and this technology allows genetically superior males to produce more offspring than would be possible through conventional mating. Conventional AI has been enhanced in recent years with the use of sperm cells that have been flow cytometrically sorted into populations of X chromosome- and Y chromosome-bearing spermatozoa, facilitating production of offspring of a pre-determined genetic sex. A related genetic improvement technology, embryo transfer, involves non-surgical recovery of preimplantation embryos from one female and subsequent transfer into recipient females to enable genetically superior females to produce a greater number of offspring than would be possible through traditional mating. With the advent of ultrasound-guided procedures for recovery of oocytes from living females, coupled with advances in in vitro oocyte maturation, in vitro fertilization and in vitro embryo culture, the production of embryos in vitro is rapidly growing and may soon generate more embryos than are derived in vivo. Embryo micromanipulation procedures such as embryo bisection, microfertilization, and assisted embryo hatching are providing additional tools for reproductive biologists to produce live offspring. Embryo biopsy is permitting preimplantation genetic diagnosis not only for genetic sex determination but also for identification of embryonic genetic defects. Embryo culture and embryo coculture systems have also enabled proliferation of novel assisted reproductive technologies such as oocyte reconstruction and nuclear transfer.

SummaryA total of 77 embryo collection attempts from 28 donor mares resulted in 50 embryos (64.9 per cent recovery rate) ranging from Day‐6 morulae to Day‐7 expanded blastocysts. Thirty‐five of these embryos, classified as quality grades 1 and 2, were allotted randomly by developmental stage (Stage‐I, precapsulate and Stage‐II, postcapsulate) to a control group transferred non‐surgically within 1 h to synchronised recipients, and a treated group, cultured on a monolayer of foetal uterine fibroblast cells for 24 h prior to non‐surgical transfer to recipient mares. There was no significant difference in pregnancy rates between Stage‐I and Stage‐II embryos, nor between the control group (53.3 per cent) and the 24 h co‐cultured group (42.1 per cent). The use of a co‐culture system may provide a viable alternative to cryopreservation for the transport of equine embryos from one station to another.

During 1983, 887 fine wool rams were subjected to breeding soundness evaluation to determine effects of age and scrotal circumference on semen characteristics. Of rams evaluated, 94.4, 84.8, and 81.9% of young (=1.5 yr), mature (2 to 5 yr), and old (>6 yr), respectively, were rated as satis-factory. Old rams had fewer (P < 0.05) motile cells and more (P < 0.05) abnormal cells than young rams. Among older rams there was a higher percentage (P < 0.05) with testicular lesions than among young or mature rams. Scrotal circumference was positively correlated (P < 0.01) with semen volume and percentage of motile normal cells. Motility was negatively correlated (P < 0.01) with percentage of abnormal cells. Mature and old rams with large (>/=36.7 cm) testis had more (P < 0.10) abnormal cells than those in the same age groups with smaller testis. Scrotal circumference was positively correlated (P < 0.10) to volume and motility in mature and old rams, while motility was negatively correlated (P < 0.01) to percentage of abnormal cells in all age groups. Previous semen testing reduced (P < 0.05) the percentage of mature rams with leucocytes. Vaccination against epididymitis reduced (P < 0.05) the incidence of mature rams with leucocytes and testicular lesions. Brucella ovis was recovered from 54 (67.5%) of 80 ejaculates cultured. Among rams infected with B . ovis , only three (5.6%) were vaccinated against B . ovis and had their semen tested previously.