Piglets produced from in vivo blastocysts vitrified using the Cryologic Vitrification Method (solid surface vitrification) and a sealed storage container

The objective was to develop a simple successful porcine cryopreservation protocol that prevented contact between embryos and liquid nitrogen, avoiding potential contamination risks. In vivo-derived blastocysts were collected surgically from donor pigs, and two porcine embryo vitrification protocols (one used centrifugation to polarize intracytoplasmic lipids, whereas the other did not) were compared using the Cryologic Vitrification Method (CVM), which used solid surface vitrification. The CVM allowed embryos to be vitrified, without any contact between embryos and liquid nitrogen. Both protocols resulted in similar in vitro survival rates (90% and 94%) and cell number (89 ± 5 and 99 ± 5) after 48 h in vitro culture of vitrified and warmed blastocysts. The protocol that did not use centrifugation was selected for continued use. To protect vitrified embryos from contact with liquid nitrogen and potential contamination during storage, a sealed outer container was developed. Use of this sealed outer container did not affect in vitro survival of cryopreserved blastocysts. In vivo blastocysts (n = 151) were collected, vitrified, and stored using the selected protocol and sealed container. These embryos were subsequently warmed and transferred to six recipients; five became pregnant and farrowed a total of 26 piglets. This embryo vitrification method allowed porcine embryos to be successfully vitrified and stored without any contact with liquid nitrogen.     

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

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

In vivo and in vitro survival of goat embryos after freezing with ethylene glycol or glycerol

The aim of the present study was to compare the survival rates of goat morulae and blastocysts after different freezing procedures. The viability of frozen-thawed embryos was assessed both in vivo and in vitro. Two cryoprotectants, ethylene glycol and glycerol, were used and three cryoprotectant removal procedures were compared: progressive dilution in 1.0, 0.5, 0.3 and 0 M of cryoprotectant in PBS; a similar progressive dilution with cryoprotectant in PBS plus 0.25 M of sucrose; or one-step transfer in PBS containing 0.25 M of sucrose. In vitro development of frozen-thawed blastocysts was always higher than that of frozen morulae irrespective of the cryoprotectant (52 129 = 40.3% vs 23 161 = 14.3% ; P< 0.001). In vivo, however, frozen-thawed morulae developed equally as well as blastocysts after an identical freezing-thawing protocol. Development both in vivo and in vitro showed ethylene glycol to be a better cryoprotectant than glycerol for goat embryos at both developmental stages (23 vs 0%, 45 vs 35% in vitro; 34.5 vs 21%, 35 vs 23% in vivo for morulae and blastocysts, respectively).     

In vitro survival of murine morulae after quick freezing in the presence of chemically defined macromolecules and different cryoprotectants

We studied the ability of frozen-thawed mouse morulae to develop in vitro when the cryoprotectant proteins were substituted with one of the following nonorganic macromolecules: polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), and ficoll. We also determined how these agents interacted with 3 different cryoprotectants: glycerol (GLY), propylene glycol (PG), and ethylene glycol (EG). The influence of both of the above factors was measured on the basis of post-thaw morphological appearance, the percentage of development to the expanded blastocyst stage and the total cell count. Morulae (n=950) were collected from superovulated mice. Those classified as good or excellent were distributed among the 12 different freezing solutions, obtained by combining the 3 cryoprotectants with the 4 macromolecules (the 3 mentioned above, plus a control of 5% fetal calf serum) in phosphate buffered saline (PBS). Embryos frozen in PVA, PVP and ficoll tended to be a little difficult to recover from the straws. Development to the expanded blastocyst stage was significantly lower (P<0.05) in propylene glycol (43.6%) than in ethylene glycol (79.5%) or in glycerol (76.1%). Polyvinyl alcohol provided a higher survival rate when combined with glycerol (90.3) or ethylene glycol (95.0), but when it was combined with propylene glycol, only 56.5% of embryos survived after thawing. A positive interaction was observed between glycerol and PVA and between ethylene glycol and PVA or ficoll. The results indicate that fetal serum could be successfully substituted for any of the 3 chemically defined macromolecules. However, our findings also suggest that the use of PG as a cryoprotectant should be avoided when mouse morulae are frozen using the quick freezing method.     

Influence of vitrification techniques and solutions on the morphology and survival of preantral follicles after in vitro culture of caprine ovarian tissue

The objective was to compare the efficiency of various vitrification techniques and solutions for preserving morphology and viability of preantral caprine follicles enclosed in ovarian tissue. Fragments of ovarian cortex were cryopreserved by conventional vitrification (CV) in French straws, vitrification in macrotubes (MTV), or solid-surface vitrification (SSV). Six solutions containing 6 M ethylene glycol, with or without sucrose (SUC; 0.25 or 0.50 M) and/or 10% fetal calf serum (FCS) were tested (Experiment I). After 1 wk, samples were warmed and preantral follicles were examined histologically. To evaluate follicular viability (Experiment II), ovarian fragments were vitrified with the three techniques listed above, in a solution containing 0.25 M SUC and 10% FCS. After warming, follicles were assessed by the trypan blue dye exclusion test. In Experiment III, preantral follicles enclosed in ovarian tissue were vitrified using the protocol which yielded the highest percentage of viable preantral follicles (SSV with 0.25 M SUC and 10% SFB). After warming, the preantral follicles enclosed in ovarian tissue were cultured in vitro and then, were analyzed by histology and fluorescence microscopy (calcein-AM and ethidium homodimer-1). Every vitrification protocol significantly reduced the percentages of morphologically normal follicles relative to the control (88.0%); however, the addition of 0.25 M SUC and 10% FCS to the vitrification solution improved preservation of follicular morphology (67.4, 67.4, and 72.0% for CV, MTV, and SSV, respectively). Although follicular viability after SSV (80.7%) did not differ from that in fresh (non-vitrified) ovarian tissues (88.0%), after in vitro culture, percentages of viable follicles were significantly reduced (70.0%). Percentages of morphologically normal follicles after in vitro culture of vitrified ovarian tissue were similar (76.0%) to those in ovarian cortex fragments cultured without previous vitrification (83.2%). In conclusion, SSV using a solution containing 0.25 M SUC and 10% FCS, was the most efficient method for vitrifying caprine ovarian tissue.     

Effect of ovarian cystic or haemorrhagic follicles on embryo recovery and survival after transfer in hCG-ovulated rabbits.

The relationship between the presence of cystic and/or haemorrhagic follicles in both donor and recipient does and survival at birth of frozen-thawed embryos (778 embryos transferred) from 3 selected rabbit strains (NZ: New Zealand white; SY and SB: synthetic breeds) were studied. Donor does (SY:108; NZ:99; SB:96) were mated and treated with 25 IU of hCG. Only morphologically normal oviductal morulae (64-66 h) were frozen. Frozen-thawed embryos from each of the 90 donor does were transferred into the oviducts of synchronized recipient does of the same strain 48 h after injection of 25 IU of hCG (SY:31; NZ:28; SB:31). The frequency of follicular anomalies (36 and 43%) was high in both donor and recipient does, respectively, and it was not affected by strain or parity. The follicular anomalies had a negative effect on the percentage of embryos recovered in the oviduct (70 vs 77%) but not on the percentage of recovered embryos catalogued as morphologically normal (97%). The absence of follicular anomalies in recipient does had a significantly favourable effect on the pregnancy rate (63 vs 18%; P less than 0.05) and consequently on embryo survival rate at birth (26 vs 7%; P less than 0.01).     

Survival and growth of goat primordial follicles after in vitro culture of ovarian cortical slices in media containing coconut water

The development of culture systems to support the initiation of growth of primordial follicles is important to the study of the factors that control the earliest stages of folliculogenesis. We investigated the effectiveness of five culture media, two supplements and three culture periods on the survival and growth of goat primordial follicles after culturing ovarian cortex. The media were based on minimal essential minimum (MEM) and coconut water solution (CWS) added in the proportion of 0, 25, 50, 75 or 100%. The two supplements were none versus supplemented with insulin-transferrin-selenium, pyruvate, glutamine, hypoxanthine, and BSA. Pieces of goat ovarian cortex were cultured in the media for 1, 3 or 5 days and representative samples were evaluated at day 0 as non-cultured controls. The replicates were the two ovaries of five mixed breed goats. The number of primordial, intermediate, primary and secondary follicles at each period of culture and the number of degenerated follicles were evaluated. Mitotic activity of granulosa cells was studied by immunolocalization of proliferating cell nuclear antigen (PCNA). The number of follicles in each stage and degenerated follicles were statistically analyzed by ANOVA using a factorial design and the significance of differences assessed using Tukey test. Chi-square test was used to compare the percentage of follicles with PCNA positive granulosa cells. As the culture period progressed, the number of primordial follicles fell and there was a significant increase in the number of primary follicles. The fall in the number of primordial follicles was particularly marked after 1 day culture. No effect of media on the number of primordial and primary follicles was observed after culture, but MEM as well as supplements increased the number of intermediate follicles. Follicular degeneration was kept at the same level after culture in the media tested, except for pure CWS that increased the number of degenerated follicles. In contrast, addition of supplements to culture media reduced follicular degeneration. In non-cultured tissue, PCNA was expressed in granulosa cells of 31.6% of the growing follicles. This percentage had not significantly changed after 5 days culture in the various media, indicating the maintenance of proliferation activity of granulosa cells during culture. In conclusion, it is shown that goat primordial follicles may be successfully activated after in vitro culture in all media tested. However, when pure CWS is used the follicular degeneration is enhanced, but the addition of supplements to culture media decrease follicular degeneration.     

Relationship between the cell surface hydrophobicity and survival of bacteria <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> after exposures to ethanol,freezing or freeze-drying

An inverse linear relationship (P < 0.01) was detected between the cell surface hydrophobicity (CSH) and survival of ethanologenic bacteria Zymomonas mobilis 113S exposed to elevated (2.55 M) ethanol concentration. In the same way, viable cell counts of relatively hydrophobic Z. mobilis were less diminished by growing (0.85-3.40 M) ethanol concentrations as compared to more hydrophobic bacteria. Very similar inverse relationships (P < 0.01) were observed between the CSH of intact Z. mobilis and survival of cells subjected to subsequent freeze-drying or freezing/thawing cycles thereby affinity substantially lowered ability of hydrophobic bacteria to survive under adverse environments. Observed relationships were supported by significant correlations between independent analytical data of the carbohydrate content within fractions of lipopolysaccharide and surface proteins extracted from cells of varied hydrophobicity. The results suggest that the CSH could be of value to predict the ability of intact bacteria to endure stress conditions and should be monitored towards lower values during cultivation in order to reduce subsequent unwanted structural and physiological disturbances provoked by multiple stress factors.     

Developmental competence of in vitro-fertilized porcine oocytes after in vitro maturation and solid surface vitrification: effect of cryopreservation on oocyte antioxidative system and cell cycle stage

The susceptibility of in vitro matured (IVM) porcine oocytes to be fertilized in vitro after vitrification was investigated. IVM oocytes were cryopreserved by solid surface vitrification (SSV) or treated with cryoprotectants (toxicity control, TC). Control oocytes were not treated or vitrified. Live oocytes in the three groups were in vitro fertilized (IVF) and then cultured (IVC) for 6 days. In vitro maturation and IVC were performed under 5% or 20% O(2) tension. The percentage of live oocytes in the SSV group was lower than those in the control and TC groups. Fertilization rates after SSV were significantly lower than in the control group. Significantly fewer penetrated oocytes formed male pronuclei in the SSV group than in the control and TC groups. Cleavage rates were significantly lower in the SSV group than in the control and TC groups. Blastocyst formation rates in the control and TC groups were similar, whereas only a single embryo developed to the blastocyst stage from 113 oocytes after vitrification. Blastocyst formation rates in the control group and in the TC group were significantly higher under 5% O(2) IVC than under 20% O(2) IVC. Oxygen tension during IVM had no effect on embryo development. The glutathione (GSH) content of vitrified oocytes was significantly lower than in the controls. In contrast, the H(2)O(2) level was higher in vitrified oocytes than in control oocytes. Vitrification caused parthenogenetic activation in 44.9% of unfertilized oocytes. This significant increase in parthenogenetic activation along with significantly dropped GSH level in vitrified oocytes may explain the decreased ability of the SSV group to form male pronuclei. These factors might have contributed to the poor developmental competence of vitrified oocytes.     

Pre-treatment neutrophil to lymphocyte ratio is elevated in epithelial ovarian cancer and predicts survival after treatment

Purpose  Inflammatory cells can both suppress and stimulate tumor growth, and the influence of inflammatory cells on clinical outcome has been the focus of many studies. The purpose of this study was to evaluate the effectiveness of the neutrophil to lymphocyte ratio (NLR), a measure of the systemic inflammatory response, as an additional discriminative biomarker in epithelial ovarian cancer and to determine whether it predicts survival and recurrence. Methods  We studied 192 patients with epithelial ovarian cancer, 173 with benign ovarian tumors, 229 with benign gynecologic disease, and 405 healthy controls. Serum CA125 levels and leukocyte counts according to subtypes were recorded prior to treatment in all study subjects. In epithelial ovarian cancer, the diagnostic usefulness of NLR, in combination with CA125, was evaluated. The correlation between NLR and overall and disease-free survival was analyzed using both univariate and multivariate analyses adjusting for the known prognostic factors (age, stage, cell type, and grade). Results  Preoperative NLR in ovarian cancer subjects (mean 6.02) was significantly higher than that in benign ovarian tumor subjects (mean 2.57), benign gynecologic disease subjects (mean 2.55), and healthy controls (mean 1.98) (P < 0.001). The sensitivity and specificity of NLR in detecting ovarian cancer was 66.1% (95% CI, 59.52–72.68%) and 82.7% (95% CI, 79.02–86.38%), respectively (cutoff value: 2.60). In early stage ovarian cancer, CA125 was not elevated in 19 out of 49 patients. Seven (36.8%) of these 19 patients were NLR positive. On Cox multivariate analysis, NLR positive, stage III/IV, and older age were independent poor prognostic factors, and being NLR positive was the most powerful predictive variable (Hazard Ratio = 8.42 [95% CI: 1.09–64.84], P = 0.041). Conclusions  Our findings provide evidence for the association between NLR and epithelial ovarian cancer. Preoperative NLR, in combination with CA125, may represent a simple and cost-effective method of identifying ovarian cancers, and an elevated NLR may predict an adverse outcome in ovarian cancer.     

The survival of mouse oocytes shows little or no correlation with the vitrification or freezing of the external medium,but the ability of the medium to vitrify is affected by its solute concentration and by the cooling rate

As survival of mouse oocytes subjected to vitrification depends far more on the warming rate than on the cooling rate, we wished to determine whether the lack of correlation between survival and cooling rate was mirrored by a lack of correlation between cooling rate and vitrification of the medium (EAFS), and between survival and the vitrification of the medium. The morphological and functional survival of the oocytes showed little or no relation to whether or not the EAFS medium vitrified or froze. We studied if the droplet size and the elapsed time (between placing the droplet on the Cryotop and the start of cooling) affects the result through modification of the cooling rate and solute concentration. Dehydration was rapid; consequently, the time between the placing the droplets into a Cryotop and cooling must be held to a minimum. The size of the EAFS droplet that is being cooled does not seem to affect vitrification. Finally, the degree to which samples of EAFS vitrify is firmly dependent on both its solute concentration and the cooling rate.     

Effect of addition of hyaluronan to embryo culture medium on survival of bovine embryos in vitro following vitrification and establishment of pregnancy after transfer to recipients

Two experiments were conducted to determine whether addition of hyaluronan to culture medium could improve survival of bovine embryos after vitrification or following embryo transfer. In Experiment 1, embryos were produced in vitro and cultured for 7 days in modified synthetic oviductal fluid (SOF) containing one of four concentrations of hyaluronan (0, 0.1, 0.5, or 1 mg/mL), with or without 4 mg/mL of bovine serum albumin (BSA). On Day 7 after insemination, blastocysts and expanded blastocysts were vitrified using open-pulled straws. At a concentration of 1 mg/mL, hyaluronan increased (P < 0.05) the percentage of oocytes that were blastocysts and re-expansion rate at 24 h after warming. At 0.5 mg/mL, hyaluronan tended (P < 0.10) to increase re-expansion rate at 48 h after warming and increased (P < 0.05) embryo hatching rate at 24 and 72 h. Treatment with BSA caused a slight reduction in cleavage rate (P < 0.05), but only for cultures containing hyaluronan (BSA × hyaluronan, P = 0.10), an increase in the percentage of oocytes that became blastocysts (P < 0.001), and a reduction in re-expansion rates (P < 0.001) and hatching rates (P < 0.05 or P < 0.01) at all times examined. In Experiment 2, embryos were produced in vitro and cultured in modified SOF containing 4 mg/mL BSA, with or without 1 mg/mL hyaluronan. At 159-162 h after insemination, grade 1 morula, blastocysts and expanded blastocysts were harvested for embryo transfer. Harvested embryos were transferred individually to lactating Holstein recipients with a palpable corpus luteum on Day 7 after presumptive ovulation. There was an interaction (P < 0.05) between hyaluronan and embryo stage on pregnancy rate. Recipients that received morula and blastocyst stage embryos treated with hyaluronan had a higher pregnancy rate than recipients that received control embryos of the same stage. There was no effect of hyaluronan on pregnancy rates of recipients that received expanded blastocysts. In conclusion, addition of hyaluronan to embryo culture enhanced blastocyst yield, improved survival following vitrification, and enhanced the post-transfer survival of fresh morula and blastocyst stage embryos.     

Immunotherapy with chimeric NKG2D receptors leads to long-term tumor-free survival and development of host antitumor immunity in murine ovarian cancer

Ovarian cancer is one of the leading causes of cancer death in women and the development of novel therapies is needed to complement the standard treatment options such as chemotherapy and radiation. In this study, we show that treatment with T cells expressing a chimeric NKG2D receptor (chNKG2D) was able to lead to long-term, tumor-free survival in mice bearing established ovarian tumors. Tumor-free mice were able to reject a rechallenge with ovarian tumor cells 225 days after original tumor injection. In addition, chNKG2D T cell treatment induced specific host immune responses to ovarian tumor cells, including the development of both CD8+ and CD4+ T cell tumor-specific memory responses. The chNKG2D T cells reduced the ovarian tumor burden using both cytotoxic and cytokine-dependent pathways. Specifically, chNKG2D T cell expression of perforin, GM-CSF, and IFN-gamma were essential for complete antitumor efficacy.     

Diffusion of lactate and ammonium in relation to growth of Geotrichum candidum at the surface of solid media

Geotrichum candidum was cultivated at the surface of solid model media containing peptone to simulate the composition of Camembert cheese. The surface growth of G. candidum induced the diffusion of substrates from the core to the rind and the diffusion of produced metabolites from the rind to the core. In the range of pH measured during G. candidum growth, constant diffusion coefficients were found for lactate and ammonium, 0.4 and 0.8 cm(2) day(-1), respectively, determined in sterile culture medium. Growth kinetics are described using the Verlhust model and both lactate consumption and ammonium production are considered as partially linked to growth. The experimental diffusion gradients of lactate and ammonium recorded during G. candidum growth have been fitted. The diffusion/reaction model was found to match with experimental data until the end of growth, except with regard to ammonium concentration gradients in the presence of lactate in the medium. Indeed, G. candidum preferentially assimilated peptone over lactate as a carbon source, resulting in an almost cessation of ammonium release before the end of growth. On peptone, it was found that the proton transfer did not account for the ammonium concentration gradients. Indeed, amino acids, being positively charged, are involved in the proton transfer at the beginning of growth. This effect can be neglected in the presence of lactate within the medium, and the sum of both lactate consumption and ammonium release gradients corresponded well to the proton transfer gradients, confirming that both components are responsible for the pH increase observed during the ripening of soft Camembert cheese.     

低氟铝长期与高氟铝短期暴露后大鼠骨生长的比较研究

目的观察不同剂量氟铝联合摄入时间长短对大鼠纵向骨生长及骨代谢的影响。方法48只8周龄体重170~190g清洁级SD大鼠,随机分成正常对照,低氟铝和高氟铝组,且分别设45d和90d组。进行胫骨近端生长板和干骺端松质骨的骨形态计量学分析。结果与正常组比较,高氟铝组生长板增厚,45d组软骨细胞层次清楚,排列整齐,形态无异常,而90d组软骨细胞拥挤,潴留;低氟铝(45d和90d)组干骺端次级小梁骨矿化周长、骨形成率、成骨细胞周长都增加,且骨吸收周长在90d组增加;上述骨代谢指标在高氟铝45d组均增加,90d组均降低。结论高氟铝短期暴露刺激软骨生长,长期抑制纵向骨生长。低氟铝短期暴露只增加次级小梁骨形成,低氟铝长期与高氟铝短期暴露均可刺激小梁骨形成,增加骨吸收,高氟铝长期抑制骨形成和吸收。     

Characteristics of DNA synthesis in Chinese hamster cells in exponential and stationary culture growth stages after cell exposure to elevated temperature

Hyperthermia (43 degrees C for 1 hour) is shown to induce a reduction of DNA synthesis and a change in molecular weight distributions in the pool of newly synthesized DNA in cultured Chinese hamster cells. The DNA synthesis inhibition extent as well as the character of disturbances in DNA replication are different in cells of the exponential and the stationary cultures.     

Resynchronization of ovulation and timed insemination in lactating dairy cows, II: assigning protocols according to stages of the estrous cycle, or presence of ovarian cysts or anestrus

Pregnancy rates were compared in lactating dairy cows (n = 1083) assigned to protocols for resynchronization of ovulation based on stages of the estrous cycle, or presence of ovarian cysts or anestrus. Cows were detected not pregnant by ultrasonography 30 d after a previous AI (study day 0) and classified as diestrus, metestrus, proestrus, with ovarian cysts or anestrus. Cows in diestrus (January-May) were assigned to either Ovsynch (GnRH day 0, PGF2alpha day 7, GnRH day 9, and timed-AI [TAI] 16 h later; n = 96), or Quicksynch (PGF2alpha day 0, estradiol cypionate [ECP] day 1, AI at detected estrus [AIDE] on day 2, or TAI on day 3; n = 96). Cows in diestrus (June-December) were assigned to either Ovsynch (n = 156) or Modified Quicksynch (PGF2alpha day 0, ECP day 1, AIDE days 2 and 3, and to Ovsynch on day 4 if not detected in estrus; n = 142). Cows in metestrus were assigned either to Ovsynch (n = 68), Heatsynch (GnRH day 0, PGF2alpha day 7, ECP day 8, AIDE day 9, or TAI day 10; n = 62), or GnRH + Ovsynch (GnRH on day 0, followed by Ovsynch on day 8; n = 64). Cows in proestrus, with ovarian cysts, or anestrus were assigned to either Ovsynch (proestrus n = 89, ovarian cysts n = 97, anestrus n = 8) or GnRH + Ovsynch (proestrus n = 87, ovarian cysts n = 109, anestrus n = 9). Pregnancy rate was evaluated 30, 55 and 90 d after resynchronized AI. For cows in diestrus (January-May), pregnancy rates were higher for Ovsynch (35.9, 29.2 and 26.0%) than for Quicksynch (21.7, 16.7 and 15.6%). For cows in diestrus (June-December), pregnancy rates were similar for Ovsynch (34.4, 24.0 and 23.6%) and Modified Quicksynch (27.1, 26.2 and 21.6%). For cows in metestrus, pregnancy rates were higher for GnRH + Ovsynch (33.3, 24.5 and 20.3%) than for Heatsynch (20.3, 12.9 and 9.8%). For cows with ovarian cysts, pregnancy rates were higher for GnRH + Ovsynch (30.3, 26.6 and 22.9%) than for Ovsynch (20.2, 18.5 and 14.7%). Assignment to resynchronization protocols based on the stages of the estrous cycle, or presence of ovarian cysts improved pregnancy rates.