Day Of Insemination Research Articles

Pre-washed frozen donor sperm is associated with lower pregnancy rates compared to semen processed immediately prior to donor insemination.

Objective: Pre-washed frozen sperm has recently been promoted by some commercial sperm banks. The obvious benefit is reduced need for laboratory resources for physicians wishing to perform donor intrauterine insemination (IUI-D). Although many physicians would consider this a major advantage, comparison to outcome using traditional semen cryopreservation (unwashed) and post-thaw processing is very limited. The purpose of this study is to compare sperm motility parameters and cycle outcome in women undergoing intrauterine insemination (IUI) with either prewashed donor sperm or unwashed donor sperm requiring post thaw preparation. Design: Retrospective analysis of women undergoing LH timed IUI-D and FSH stimulated IUI-D with pre-washed or unwashed frozen donor sperm from January 1, 1999 through January 31, 2001. Materials/Methods: Patients undergoing donor insemination using sperm from a commercial bank (Cryobiology–Ohio) ordered pre-washed or unwashed frozen based primarily on availability. Pre-washed specimens were centrifuged over a density gradient and re-suspended in a solution of test-yolk buffer (TYB) to achieve a desired density of motile sperm prior to cryopreservation. Unwashed semen samples were diluted in TYB and centrifuged and resuspended as necessary (no density gradient) to achieve a desired density of motile sperm prior to freeze. Pre-washed specimens were thawed immediately prior to insemination requiring no further preparation. Unwashed specimens were thawed on the day of insemination, and processed via centrifugation over a density gradient just prior to IUI. All specimens were analyzed by computer assisted semen analyzer (CASA–IVOS/Hamilton Thorne) post thaw. Unwashed specimens were additionally analyzed following processing and prior to IUI. Results: Please refer to table. Results. ∗Type of cycle:Timed IUIFSH IUIType of vial:Unwashed vialsPre-washed vialsUnwashed vialsPre-washed vialsTotal number cycles:15178125Average age:32.8534.1432.7536.20Mean total progressive (M):7.868.7910.2310.14Mean progressive motility:37.97%33.11%44.63%36.06%Positive hCG:401.141Percent positive hCG:26.49%∗p < 0.5T (Timed IUI: Unwashed vs. Prewashed; Fisher’s Exact).14.10%33.33%20.00%∗ p < 0.5T (Timed IUI: Unwashed vs. Prewashed; Fisher’s Exact). Open table in a new tab Conclusions: Pre-washed semen had relatively lower percent progressive sperm motility than sperm processed post-thaw. The pregnancy rates following insemination with pre-washed sperm was markedly lower than the rates following insemination with sperm processed post thaw. Although the use of pre-washed sperm inarguably allows IUI-D to be performed by physicians with limited access to reproductive laboratories, this may come at the cost of lower fecundity. Further refinement of sperm pre-washing techniques may ultimately lead to improved outcome. At the present time, physicians with limited access to reproductive laboratories may wish to consider other alternatives to IUI with pre-washed donor sperm.

A hipertermia durante o estro pode afetar o desempenho reprodutivo de fêmeas suínas

O objetivo deste estudo foi avaliar o efeito da hipertermia no dia da inseminação artificial (IA) até o 13º dia de gestação, sobre o desempenho reprodutivo de leitoas e porcas. No experimento 1, as 488 fêmeas foram divididas em dois grupos de acordo com a temperatura retal no dia da primeira inseminação artificial (IA), respectivamente, G1 = £39,5ºC (normotermia) e G2 = &gt;39,5ºC (hipertermia). As fêmeas com hipertermia apresentaram taxas de retorno ao estro (TRE) maiores e taxas de parto (TP) e tamanho de leitegada (TL) menores (p&lt;0,05), quando comparadas às fêmeas com normotermia. No experimento 2, as 764 fêmeas foram classificadas em quatro grupos, respectivamente, G1 = hipertermia no dia da IA; G2 = hipertermia em um dos primeiros 4 dias após a IA; G3 = hipertermia em pelo menos um dia nos dois períodos anteriores e G4 = hipertermia do 10º ao 13º dia de gestação. Fêmeas com hipertermia no dia da primeira IA e no período inicial de gestação apresentaram maior TRE e menor TP no G1 e G2 (p£ 0,01) e o TL foi menor (p&lt;0,01) nos grupos G1, G2 e G3, quando comparado às fêmeas com normotermia. Na fase de ligação embrio-maternal (G4), não foram observadas diferenças entre as fêmeas com normotermia e hipertermia. O experimento 3, no qual foram acompanhadas 102 leitoas, das quais 88 foram abatidas aos 29-34 dias de gestação, mostrou uma maior taxa de retorno ao estro (TRE) (p£ 0,01) e menor taxa de prenhez (TPr) (p£ 0,05), além de um menor número médio de embriões viáveis (NEV) e menor taxa de sobrevivência embrionária (SE) (p&lt;0,05), nas leitoas com hipertermia. Com base nesses resultados, pode-se concluir que fêmeas com hipertermia no dia da IA e nos primeiros quatro dias após, podem apresentar prejuízos para o desempenho reprodutivo.

Effect of short-term treatment with bovine somatotropin at estrus on conception rate and luteal function of repeat-breeding dairy cows

We studied the effect of recombinant bovine somatotropin (rbST) at the time of estrus on progesterone concentrations and conception rates of repeat-breeding Holstein cows. We used repeat-breeding cows of varied parity (n=510). All the animals were clinically healthy and had had at least three unsuccessful services before entering the study. After detection of estrus, the cows were randomly assigned to either a treated (n = 201) or a control (n = 309) group. The animals in the treated group were given rbST (500 mg sc) at the time of estrus and again 10 d later. Artificial insemination was performed 12 h after the first detection of estrus. In order to evaluate the effect of rbST on luteal function, blood samples were taken from 10 cows in each group every 3 d for 18 d, starting on the day of insemination (Day 0) to determine progesterone concentrations. Conception rates were significantly higher (P < 0.05) in the cows treated with rbST (29.3%) than in the control cows (16.9%). The effects of rbST were maximal in cows with 8 or more previous unsuccessful services and in cows with 2 to 4 calvings. Progesterone concentrations tended to be higher in nonpregnant cows that were treated with rbST than in those that were not treated. The difference between groups was significant (P < 0.05) on Day 18 after insemination. In pregnant cows there were no significant differences in progesterone concentrations between treated and nontreated animals at any time. Treatment with rbST at estrus improved the conception rate of repeat-breeding Holstein cows. This effect was associated with an increase in circulating progesterone concentrations on Day 18.

Effect of the in vitro culture system on the kinetics of blastocyst development and sex ratio of bovine embryos

Bovine blastocysts were produced using 6 different systems: 5 commonly used in vitro culture systems (synthetic oviduct fluid medium - SOF - without fetal calf serum, SOF supplemented with 10% serum for the entire culture period, SOF supplemented with 10% serum from Day 4 of culture, M199 coculture with bovine oviduct epithelial cells, M199 coculture with granulosa cell monolayer) and 1 in vivo culture system involving collection of blastocysts from superovulated bovine donors at Day 7. Zygotes obtained from IVM/IVF were assigned randomly to 1 of the 5 systems tested and were cultured for 9 d( Day 0= day of insemination). Cleavage, development to the blastocyst stage and blastocyst sex ratio were assessed in all treatments. In addition, the effect of the IVC system on the kinetics of blastocyst development and sex ratio was assessed on Days 6, 7, 8, and 9. The presence of fetal calf serum in SOF not only resulted in faster development (19.1% of blastocysts in SOF supplemented with serum vs 7.1% in absence of serum at Day 6; P < 0.05) and increased blastocyst production (47.5% of blastocysts in SOF supplemented with serum vs 34.4% in absence of serum; P < 0.05) but it also enhanced overall male survival. The coculture systems produced fewer blastocysts than culture in SOF (27.6 to 28.3% in coculture vs 47.5% in SOF supplemented with serum; P < 0.05), but similar to SOF without fetal calf serum, they had no effect on blastocyst sex ratio.

Gel-Loading Tip As Container for Vitrification of In Vitro-Produced Bovine Embryos.

Recently, new vitrification methods for bovine embryos characterized by ultra-rapid cooling have been reported. The present study was undertaken to determine the availability of gel-loading tips as containers for vitrification of in vitro-produced bovine embryos at various developmental stages. Bovine embryos at Days-1, -2, -3, -4, -5, and -7 (the day of insemination was defined as Day-0) were placed in 0.6~0.7 μl of vitrification solution (20% ethylene glycol, 20% DMSO and 0.6 M sucrose) in a commercially available gel-loading tip, and cooled by immersing in liquid nitrogen. Survival of the vitrified embryos was evaluated by in vitro development into the blastocyst stage. There was no significant difference (P ≥0.05) between the survival rates for fresh and vitrified embryos at Day-1 (70.5 and 58.0%, respectively), Day-2 (75.9 and 68.3%, respectively), Day-3 (75.9 and 57.9%, respectively), Day-4 (80.0 and 67.6%, respectively), Day-5 (82.1 and 60.0%, respectively) and Day-7 (100 and 97.8%, respectively). And no significant differences were found between the total numbers of cells or the proportion of inner cell mass cells of blastocysts developed from fresh versus vitrified embryos. These results demonstrate that the gel-loading tip is applicable as a container for vitrification of in vitro-produced bovine embryos at the 2-cell to blastocyst stages.

Relationship between maternal endocrine environment, early embryo development and inhibition of the luteolytic mechanism in cows

In this study, the relationship between maternal hormone environment and early embryo development in mature non-lactating Holstein-Friesian cows was investigated. Animals were inseminated at either 72 or 96 h after prostaglandin injection (n = 23) or were left as uninseminated controls (n = 10). Plasma samples were collected once a day from the first day of insemination (day 1) until day 16, when the cows underwent an oxytocin challenge, and were then slaughtered and their reproductive tracts removed. The tracts were flushed to collect embryos and the flushes were measured for interferon tau (IFN-tau) activity. Inseminated cows without an embryo on day 16 (n = 5) underwent both delayed ovulation (indicated by delayed decrease in oestradiol concentrations) and a delayed increase in progesterone concentrations after ovulation compared with cows with an embryo on day 16 (n = 15). Within the group of cows with an embryo, those with poorly developed embryos producing undetectable concentrations of IFN-tau (n = 7) had similar oestradiol profiles but underwent a delayed progesterone increase after ovulation compared with cows with well developed embryos producing measurable quantities of IFN-tau (n = 8). In the cows with an embryo, the plasma concentration of 13,14-dihydro-15-keto PGF2a, the principal metabolite of PGF2a, after injection of oxytocin was lower than that of control cows and cows without an embryo. However, when the cows with an embryo were compared on the basis of production of embryonic IFN-tau, the PGF2a response to oxytocin was attenuated completely in cows that had measurable IFN-tau activity, whereas a response of similar magnitude to that in control cows and cows without an embryo was observed in those with undetectable IFN-tau activities. In conclusion, the successful maternal recognition of pregnancy in cows depends on the presence of a sufficiently well developed embryo producing sufficient quantities of IFN-tau, which is, in turn, dependent on an appropriate pattern of maternal progesterone secretion.

The potential effect of controlled ovarian hyperstimulation on follicular vascular perfusion: a study applying power Doppler ultrasonography within a treatment programme using donor sperm

This was a prospective observational study to evaluate the effect of controlled ovarian hyperstimulation on follicular vascularity. A subjective grading system was applied to assess vascularity using transvaginal power Doppler ultrasonography on the day of insemination (32-36 hours post-HCG administration) in donor treatment cycles undergoing either controlled ovarian hyperstimulation (COH group) with gonadotrophin and intrauterine insemination or intracervical insemination with/without clomiphene stimulation (non-COH group). The incidence of follicles (81% vs. 53%; P < 0·05) as well as cycles (63% vs. 49%; P < 0·05) with uniformly high grade vascularity was significantly higher in treatments that had controlled ovarian hyperstimulation compared with unstimulated or clomiphene induced donor cycles. Pregnancy rates were higher and early pregnancy loss rates lower, in cycles with uniformly high grade follicular vascularity in both treatment groups compared with other vascularity grades. Using multiple logistic regression analysis, the only independent variable that was significantly related to pregnancy rates in this treatment programme was follicular vascularity. Controlled ovarian hyperstimulation appears to have an effect on follicular vascularity and this may contribute to improved outcome in donor insemination treatment programmes.

Preliminary observations on the concentrations of serum luteinizing hormone and follicle-stimulating hormone during gestation in bactrian camels (Camelus bactrianus).

The concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the serum of the Bactrian camel (Camelus bactrianus) throughout pregnancy were analysed by radioimmunoassay. The samples were withdrawn at intervals of half a month. The serum LH concentrations varied significantly between 2.2+/-0.9 and 20.3+/-18.8 ng/ml during the pregnancy, with the highest value on the day of artificial insemination, which indicated that a preovulatory LH peak appeared. Thereafter, the LH concentration tended to fall, reaching a fairly constant low level by the eighth month of pregnancy. The serum FSH concentrations varied between 7.0+/-0.2 and 28.9+/-0.4 ng/ml during gestation, with the peak value being reached at 4.5 months, followed by a marked drop to 7.0+/-0.2 ng/ml at 7.5 months.

New method for culture of zona-included or zona-free embryos: The Well of the Well (WOW) system

Culture of mammalian zygotes individually and in small groups results in lower developmental rates than culture of large groups. Zona-free zygotes also have impaired developmental potential in current culture systems. This paper describes a new approach to resolve the problems, the Well of the Well (WOW) system. Small wells (WOWs) were formed in four-well dishes by melting the bottom with heated steel rods. The WOWs were then rinsed, the wells were filled with medium, and the embryos were placed into the WOWs. To test the value of the WOW system a 3 × 3 factorial experiment was performed. Bovine presumptive zygotes were cultured from day 1 to day 7 (day 0: day of insemination) using three modules (single embryos, embryo groups of five, or single zona-digested embryos) and three different culture systems (400 μl medium, 200 μl drops, or WOWs). An additional control group consisted of 40 to 50 embryos cultured in 400 μl medium. The WOW system resulted in higher blastocyst/oocyte rates for all three modules (single: 59%; group of five: 61%; single zona-digested: 53%) than the culture in drops or in wells (P < 0.05 for all). The developmental rate was independent of the number of WOWs per well. The cell number of blastocysts cultured in the WOW system did not differ from that of the controls. Apart from its theoretical value in revealing the role of different factors influencing embryo development in vitro, the WOW system may have immediate practical consequences in certain areas of mammalian embryo production. Mol Reprod Dev 55:256–264, 2000. © 2000 Wiley-Liss, Inc.

New method for culture of zona-included or zona-free embryos: the Well of the Well (WOW) system.

Culture of mammalian zygotes individually and in small groups results in lower developmental rates than culture of large groups. Zona-free zygotes also have impaired developmental potential in current culture systems. This paper describes a new approach to resolve the problems, the Well of the Well (WOW) system. Small wells (WOWs) were formed in four-well dishes by melting the bottom with heated steel rods. The WOWs were then rinsed, the wells were filled with medium, and the embryos were placed into the WOWs. To test the value of the WOW system a 3 x 3 factorial experiment was performed. Bovine presumptive zygotes were cultured from day 1 to day 7 (day 0: day of insemination) using three modules (single embryos, embryo groups of five, or single zona-digested embryos) and three different culture systems (400 microl medium, 200 microl drops, or WOWs). An additional control group consisted of 40 to 50 embryos cultured in 400 microl medium. The WOW system resulted in higher blastocyst/oocyte rates for all three modules (single: 59%; group of five: 61%; single zona-digested: 53%) than the culture in drops or in wells (P < 0.05 for all). The developmental rate was independent of the number of WOWs per well. The cell number of blastocysts cultured in the WOW system did not differ from that of the controls. Apart from its theoretical value in revealing the role of different factors influencing embryo development in vitro, the WOW system may have immediate practical consequences in certain areas of mammalian embryo production.

The relationship between various animal and management factors and milk urea, and its association with reproductive performance of dairy cows grazing pasture

This study was designed to determine the use of milk urea to diagnose nutrient imbalances in diets of dairy cattle grazing pasture and infertility problems associated with excess dietary nitrogen (N). A first experiment investigated the influence of the ratio of N/water soluble carbohydrate (WSC) (g/g) and N/metabolisable energy (ME) (g/MJ) (% of estimated intake) in the diet, and animal and management factors, on urea concentrations of individual cow or bulk milk samples collected at monthly intervals over a 2-year period from a herd of 108 Friesian dairy cows grazing pasture. Mean±S.E. daily milk yield was 18.4±0.11 l milk/cow with 40.3±0.25 mg urea/dl of milk. This relatively high milk urea value is in line with the high estimated N/WSC ratio, which was three times the recommended ratio for that level of production. The ratio of N/WSC in the diet alone explained 8.8% of the variation in milk urea from individual cows, and this was increased to 21.5% when genetic merit, cow age, milk yield and milk protein content were included in the model. Level of feeding and cow live-weight had no significant effect on milk urea. Milk urea levels were lowest (at 32 mg/dl) when cows were between 40 and 60 days in lactation. Cows had higher ( P<0.001) levels of urea in their milk from January to June, when they grazed kikuyu grass pasture, than for the rest of the year when they grazed ryegrass pasture (43 vs. 35 mg/dl). There was a stronger relationship between N/WSC and the urea content of bulk milk ( r 2=30%) than individual cow milk ( r 2=11%), and this relationship was curvilinear. Substituting ME for WSC explained a further 2% and 2.7% of variation in bulk and individual milk urea, respectively. In a second experiment, the relationship between milk urea and the rates of non-return to service of Friesian cows grazing pasture was investigated. Milk samples were collected from 556 cows on four farms on the day of artificial insemination. The urea content of milk was not related ( P<0.05) to the chances of not returning to service. From the results of this study, it may be concluded that the urea content of bulk, but not individual cow, milk is a useful tool for assessing the appropriateness of the N/WSC ratio in the diet of dairy cows grazing pasture. There was no evidence that high milk urea levels are associated with poor reproductive performance in dairy cows grazing pasture.

Relationship between peripheral estrogen concentrations at insemination and subsequent fetal loss in cattle

In a survey on pregnancy rate and embryonic losses in dairy cattle on 6 Israeli farms, cows (n=78) were divided into 3 groups on the basis of ultrasonography at 21 d post insemination; pregnancy diagnosis at 40 to 50 d post insemination and blood progesterone (P 4) levels at 21 d. The groups were either pregnant (P 4 level >1.0 ng/mL); not pregnant (P 4 <0.5 ng/mL), or showed early embryo loss (P 4 >1.0 ng/mL and the presence of an embryonic vesicle on D 21 but later returned to estrus or were found not pregnant on D 40 to 50). On the day of insemination, peripheral estrogen was significantly higher (P <0.05) in the early embryo loss group (15.3±1.1 pg/mL, n=27). than in pregnant (9.4±0.6 pg/mL, n=26) or not pregnant (9.6±0.7 pg/mL, n=25) group. The cows on 3 farms which were fed 1 to 2 kg/d of vetch ( Vicia sativa ), an estrogenic legume, had higher estrogen concentrations on the day of insemination than cows (2 farms) fed other legumes (13.7±0.64, n=58 vs 10.7±0.8 pg/mL, n=42; P<0.01). On one of the 3 farms, vetch was replaced with alfalfa after the first year. Following the cessation of vetch feeding the estrogen concentrations in the blood decreased from 32±5 pg/mL to 14±2 pg/mL (n=9). These data suggest that high peripheral estrogen on the day of insemination is associated with early embryonic loss. These data also indicate that estrogen concentrations on the day of insemination can be influenced by diet.

Comparison of sex ratio and cell number of ivm-ivf bovine blastocysts co-cultured with bovine oviduct epithelial cells or with vero cells

The influence of 2 co-culture systems (BOEC and Vero cells) on the development rates, quality grades and sex ratios of IVM-IVF bovine embryos were studied. Zygotes obtained after IVF were co-cultured in each co-culture system for 7 and 8 d (Day 0= day of insemination) in B 2medium. No effect of the co-culture system was observed on development rates measured on Days 7 and 8. However, Vero cell co-culture had a positive influence on embryo quality. Irrespective of their sex, embryos produced on Vero cells showed higher cells number than those co-cultured on BOEC (103.4 ± 3.8 and 97 ± 8.12 for BOEC vs 113.7 ± 3.5 and 114 ± 5.9 for Vero cells at Days 7 and 8, respectively; P < 0.05). The percentage of male embryos was increased in the two co-culture systems (60.7% males for BOEC ; P<0.05 vs 63% males for Vero cells; P<0.01) on Day 7. In both co-culture systems the increase in the percentage of males was more obvious for embryos reaching the most advanced stage (expanded blastocysts). The results show that Vero cells improved the quality grade of bovine embryos produced in vitro, and thus are recommended for use as a safe co-culture system that does not contain pathogens.

Bovine viral diarrhea virus replication in bovine follicular epithelial cells derived from persistently infected heifers.

Bovine follicle fluid and oocytes surrounded by follicular epithelial (FE) cells were collected from ovaries of two heifers persistently infected with bovine viral diarrhea virus (BVDV). BVDV was present in the follicle fluid at a higher titer than in serum. The oocytes were matured in vitro under culture conditions of 39 degrees C in humidified air containing 5% CO2. In vitro fertilization was performed after 24 hr in culture (the day of insemination was defined as day 1), and culture was continued through day 10. BVDV was present in the culture medium at titers of 10(2.25) to 10(3.25) TC(I)D50/0.1 ml. The virus was also detected in FE cells collected on day 10. Viral antigen was demonstrated in the cytoplasm of FE cells by the indirect immunofluorescence technique. However, no BVDV was detected in the embryos on day 10. These findings suggested that the oocytes or embryos were unlikely to be infected with BVDV, but that the FE cells were infected with BVDV and supported virus replication in cattle persistently infected with BVDV.

Morphological changes of in‐vitro‐produced bovine blastocysts after vitrification, in‐straw direct rehydration, and culture

Morphological signs of injury and regeneration following vitrification and warming of bovine embryos were studied by light and electron microscopy. In-vitro-produced Day 7 expanded blastocysts (Day 0 = day of insemination) were vitrified by a two-step equilibration method using ethylene glycol and dimethyl sulphoxide as cryoprotectants. Thawing was performed by in-straw direct rehydration, followed by in vitro culture on a granulosa cell monolayer. Embryos were processed for transmission electron microscopy immediately after warming (0 hr) as well as after 4 hr or 24 hr of culture following warming. A control group of unfrozen embryos was also processed. At 0 hr after warming, except for a rapid collapse of the blastocoele, only minor changes were detectable by stereomicroscope. However, at the ultrastructural level, signs of extensive injury were seen, including a general distension or shrinkage of mitochondria, disintegration of cell adhesions between adjacent trophoblastic cells, and complete rupture of some cells. At 4 hr, stereomicroscopic investigation revealed collapsed blastocoele and a darkened granular appearance of the cell mass. At the ultrastructural level, signs of regeneration were also observable: cells with minor injuries were re-assembled in a central area forming a small blastocoele, cell adhesion structures were re-established, and damage of mitochondria was less severe. The majority of irreversibly damaged cells or cell debris was accumulated in the perivitelline space. At 24 hr, stereomicroscopic investigation of surviving blastocysts showed no signs of the previous injury. At the ultrastructural level, cellular debris in the perivitelline space and some degenerated cells in the blastocoele were the only signs of previous injuries. In conclusion, ultrastructural investigation revealed unexpectedly extensive damage followed by a rapid regeneration and reorganization of the embryonic structure. Mol. Reprod. Dev. 48:9–17, 1997. © 1997 Wiley-Liss, Inc.

Morphological changes of in-vitro-produced bovine blastocysts after vitrification, in-straw direct rehydration, and culture.

Morphological signs of injury and regeneration following vitrification and warming of bovine embryos were studied by light and electron microscopy. In-vitro-produced Day 7 expanded blastocysts (Day 0 = day of insemination) were vitrified by a two-step equilibration method using ethylene glycol and dimethyl sulphoxide as cryoprotectants. Thawing was performed by in-straw direct rehydration, followed by in vitro culture on a granulosa cell monolayer. Embryos were processed for transmission electron microscopy immediately after warming (0 hr) as well as after 4 hr or 24 hr of culture following warming. A control group of unfrozen embryos was also processed. At 0 hr after warming, except for a rapid collapse of the blastocoele, only minor changes were detectable by stereomicroscope. However, at the ultrastructural level, signs of extensive injury were seen, including a general distension or shrinkage of mitochondria, disintegration of cell adhesions between adjacent trophoblastic cells, and complete rupture of some cells. At 4 hr, stereomicroscopic investigation revealed collapsed blastocoele and a darkened granular appearance of the cell mass. At the ultrastructural level, signs of regeneration were also observable: cells with minor injuries were re-assembled in a central area forming a small blastocoele, cell adhesion structures were re-established, and damage of mitochondria was less severe. The majority of irreversibly damaged cells or cell debris was accumulated in the perivitelline space. At 24 hr, stereomicroscopic investigation of surviving blastocysts showed no signs of the previous injury. At the ultrastructural level, cellular debris in the perivitelline space and some degenerated cells in the blastocoele were the only signs of previous injuries. In conclusion, ultrastructural investigation revealed unexpectedly extensive damage followed by a rapid regeneration and reorganization of the embryonic structure.

Usefulness of polyethylene glycol for cryopreservation by vitrification of in vitro-derived bovine blastocysts.

A series of five experiments measured the high survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. The vitrification solution (designated VS) contained 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline. Embryos developed in vitro at Days 7 and 8 (Day 0 = insemination day) were exposed in one step to VS for 1 min or two steps with 10% ethylene glycol for 5 min and then VS for 1 min. In both cases, the embryos were finally cryopreserved in liquid nitrogen. After the embryos were warmed rapidly and the VS solution diluted, the survival rates were assessed by monitoring hatching rate in vitro. They were 13.0% for the one-step and 72.7% for the two-step procedures (P < 0.001). When embryos were exposed to individual solutions containing 6% (w/v) of each of 4 macromolecules (polyethylene glycol, BSA, polyvinylpyrrolidone or Ficoll) in the two-step protocol and then cryopreserved, the survival rates were 79.3, 34.8, 41.4 and 57.1%, respectively. After embryos had been exposed to the VS in two steps and then cryopreserved, there were no significant differences in survival rates when the solutions were diluted with or without sucrose. These results indicated that a vitrification solution containing polyethylene glycol can be used for cryopreservation of bovine blastocysts produced in vitro, and that a two-step addition of VS improved the in vitro survival of post-warming embryos. It was also shown to be possible to dilute post-warming embryos directly without the use of sucrose solution.

Effects of bovine serum proteins in culture medium on post-warming survival of bovine blastocysts developed in vitro

Experiments were conducted to investigate the factors affecting the survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. Zygotes were obtained by in vitro maturation and fertilization of oocytes. Embryos used in this study were developed in vitro at Day 7 and 8 (Day 0 = insemination day) in modified synthetic oviduct fluid medium supplemented with calf serum or BSA. Embryos were cryopreserved in a two-step protocol consisting of exposure to 10% ethylene glycol for 5 min, followed by the original vitrification solution (designated as VS) consisting of 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5M sucrose in phosphate-buffered saline for 1 min. After warming, embryos were cultured in modified TCM-199 for an in vitro survival assay. The highest survival rate was obtained from the warmed embryos developed at Day 7 in medium supplemented with BSA (82.6%), and there were significant differences between results with calf scrum and BSA treatment (42.4 and 70.7%, respectively; P < 0.01). However, there were no significant differences in the cell numbers of embryos among the treatments. These results suggest that the survival of embryos developed in medium with BSA is superior to that of embryos developed in medium containing calf serum, although the cell numbers of the embryos developed under both media were similar.

Relationship of clomiphene dose and patient weight to successful treatment.

Our objective was to examine the relationship between patient weight and the dose of clomiphene required for pregnancy so as to assess the validity of recommendations that the dose of clomiphene be limited to 100 mg. We retrospectively analysed the weight-dose relationship in 1681 clomiphene pregnancies and the relationship between dose and pregnancy, births, multiple births, number of pre-ovulatory follicles and endometrial thickness in 2841 cycles of clomiphene treatment, 25-250 mg, for 5 days before intrauterine insemination (IUI). Doses of clomiphene >100 mg/day were used before pregnancies in 27.4% of patients who weighed >90 kg and in 14.7% of all pregnancies. In IUI cycles, pregnancies and births, but not multiple births or abortions, were related to dose. An increase in dose from 25 to 100 mg resulted in higher pregnancy and birth rates, and in an increase in the average number of pre-ovulatory follicles > or =12 mm in diameter, from 2.0 to 2.8, with no additional increase at higher doses. Endometrial thickness and cycle day of insemination were not related to dose. We conclude that doses of clomiphene may safely be increased beyond 100 mg, and that doses > or =100 mg are required in significant numbers of patients.

Therapeutic cup insemination with cryopreserved donor sperm: prognostic value of cervical mucus score at insemination and the number of motile sperm in mucus at 24 hours

To examine the prognostic value of cervical mucus score at insemination and the number of motile sperm in mucus 24 hours after therapeutic cup insemination with cryopreserved donor sperm. Retrospective analysis. Academic tertiary medical center. One hundred thirty-eight women with confirmed bilateral tubal patency who received therapeutic cup inseminations with cryopreserved donor sperm between 1986 and 1993. All insemination cycles were monitored with serial daily urinary LH determinations with a single (n = 312) insemination or two inseminations (n = 212) performed on and/or 1 day after the day of LH surge detection. A single examiner assigned cervical mucus scores in all insemination cycles and recorded the number of motile sperm in mucus 24 hours after the first insemination in dual insemination cycles. Pregnancy rate during various cervical mucus and motile sperm scores. Ninety-one women conceived (66%) and seven of these achieved two pregnancies. The overall pregnancy rate per insemination cycle was 18.7%. Age and day of insemination were the only variables identified as having significant influence on cycle outcome. Pregnancy occurred with decreasing frequency as patient age increased and was nearly twice as likely after insemination on the day after the urinary LH surge as on the day of surge detection. Insemination the day after the urinary LH surge is superior to the day of surge detection. Cervical mucus score and the number of motile sperm in mucus 24 hours after therapeutic cup insemination with cryopreserved donor sperm do not correlate with cycle outcome.