Pre- and post-implantation losses of embryos in aging female IVCS mice

Mated female mice of the IVCS strain, aged 90 (control group), 180, 210, 240, 270, 300, 330, 360 and 420 days old, were studied for pre- and post-implantation loss of embryos. The percentage of pre-implantation loss in mice aged 90 to 210 days was 1.7/11.8 (14.4%) to 2.7/11.7 (23.1%). In mice aged 240 to 300 days it increased significantly as compared to the controls (46.5-90.2% versus 14.4%). It reached 100% in 300-days-old mice. The post-implantation loss of embryos and/or fetuses in mice aged 90 to 240 days was 1.0/10.2 (9.8%) to 2.5/9.0 (27.8%). In mice aged 270 to 300 days it increased significantly compared to the controls (100% versus 9.8%). The decrease in reproductive activity appeared first in a decrease in litter size, followed by a decrease in the number of blastocyst and implantation sites, and finally by anovulation during the process of aging in IVCS mice.     

Probable influence of ova and embryo prostaglandins in the differential ovum transport in pregnant and cycling rats.

In this study we explored the possible underlying mechanism(s) of the differential transport of unfertilized and fertilized ova in cycling and pregnant rats. The number of ova recovered from rat oviducts and uterus was not significantly different in estrus, metestrus and diestrus but dropped sharply at proestrus. When estrus rats were injected with indomethacin (10(-6)), a well known inhibitor of cyclooxygenase, delivered into both ovarian bursae, and sacrificed next day at metestrus, the number of ova in the oviduct was significantly smaller (p less than 0.025) than in controls at metestrus. On the other hand, when diestrus rats were injected with PGE1 (10(-6)) delivered into both ovarian bursae, and sacrificed next day at proestrus, no ova were found in the oviducts, and only a few of them were in the uterus. When fertilized ova were recovered from oviducts and uteri at day 4 of pregnancy (corresponding to proestrus of cycling rats) an average of 4 embryos were still found in the oviducts, proving a differential ovum transport between cycling and pregnant rats. In order to establish if there exists any ova or embryo releasing factor responsible for this difference, the prostaglandins released to the incubation medium by ovum or 3-day embryo were measured. Unfertilized ova produced significantly more PGE1 (p less than 0.05) than PGE2 or PGF2 alpha. The same pattern of PG production was observed with incubated embryos, but in this case the amount of PGE1 released was significantly higher (p less than 0.01) that the PGE1 released by unfertilized ova.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of the Juice of Lime (Citrus Aurantifolia) on Estrous Cycle and Ovulation of Sprague-Dawley Rats

ObjectiveTo determine the effect of lime juice on the estrous cycle and ovulation of cyclic female rats.MethodsTwenty-five adult female Sprague-Dawley rats were used. The study was divided into 2 experiments (I and II). In experiment I, 15 rats were randomly subclassified into 3 groups (Ia, Ib, and Ic) of 5 rats each. The estrous cycles of the rats were studied for the first 16 days to establish cyclicity, after which lime juice was administered by gastric gavage for the next 24 days. Rats in group Ia received 1 mL of undiluted lime juice, rats in group Ib received 1 mL of 50% diluted lime juice, and rats in group Ic (control animals) received only distilled water. In experiment II, 10 female rats were used and were categorized into 2 groups (IIa and IIb), with 5 rats in each group. Rats in group IIa received 1 mL of undiluted lime juice during the morning of proestrus, and those in group IIb received only distilled water on the day of proestrus. The rats were killed the next day with use of chloroform anesthesia. The upper parts of the oviducts were excised and examined under the light microscope for assessment of the number of ova shed.ResultsThere was an irregular pattern in all phases of the estrous cycle of 100% of the rats given undiluted lime juice and in 80% of those given 50% diluted lime juice. There was a significant (P = .001) reduction in the number of ova shed in rats administered undiluted lime juice in comparison with the control animals. Ovulation was partially blocked, as shown by the reduced number of ova observed in the oviducts from the rats given undiluted lime juice (5.10 ± 2.37) in comparison with the control rats (12.70 ± 1.14).ConclusionIn rats, lime juice causes irregularity of the estrous cycle, partially blocks ovulation, and may possibly compromise fertility. (Endocr Pract. 2010;16:561-565)     

In vitro fertilization in the rabbit after delayed ovum recovery

Rabbit ovum donors were superovulated with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Ova were recovered 16-17 h post-hCG from oviducts immediately after killing and from excised oviducts held in saline 30 min at 33 degrees or 38 degrees C prior to ovum recovery. In vivo-capacitated spermatozoa were used to inseminate both groups of ova. Data revealed a decrease in fertilization rates following a 30-min delay at 38 degrees C in ovum recovery. Thus, 64% (44/69 ova) were fertilized with rapid recovery, whereas 43% (39/90 ova) were fertilized following a 30-min delay. The decrease in fertilization imposed by delay in ovum recovery was apparently overcome when oviduct storage was at 33 degrees C. Under these conditions, 69% of inseminated ova were fertilized. Ova inseminated with in vitro-capacitated sperm showed a similar response to delayed ovum recovery. Embryonic development in culture of ova obtained from mated does was not affected by delay in recovery at 33 degrees or 38 degrees C provided mated does had been injected only with hCG. Ova from mated does receiving both PMSG and hCG were adversely affected by a 38 degrees C delay. The data emphasize the importance of rapid ovum recovery from oviducts and suggest the possibility of altering conditions to overcome damaging effects of delayed recovery.     

Embryo transfer in cattle: Factors affecting superovulatory response,number of transferable embryos,and length of post-treatment estrous cycles

The effect of multiple ampules of frozen semen on conception rate in superovulated Holstein heifers was studied using 3 breeding regimens (n=25): 1 ampule at 12 hr (0 hr = beginning of estrus), 3 ampules at 12 hr, and 1 ampule at each of 0, 12, and 24 hrs. There was no significant effect of breeding regimen on recovered number of ova or percentage of fertilized ova. In another project, months during which heifers were superovulated with PMSG (5–8 heifers/month) did not significantly affect rate of superovulation (number of CL). Clinical records for 173 superovulatory treatments in 150 Holstein heifers were studied to obtain preliminary information on efficacy of treatment regimens, repeatability of response within heifers, and relationships between superovulation and length of estrous cycle; where indicated, contemporary, nontreated heifers were used for comparisons. Efficacy of PMSG vs FSH treatments did not differ in number of CL or number of ova recovered, but percentage of recovered ova that were transferable was greater (P<.05) for FSH (58.3) than for PMSG (42.9). There was an intraclass correlation coefficient of 0.33 (n=37) indicating repeatability within heifers in the magnitude of response to superovulatory treatment. Mean length (days) and coefficient of variation were significantly greater for superovulatory estrous cycles (cycles during which multiple CL were present; n, 141; mean, 31.2; SE, ± 1.2; CV, 46.2%) than for contemporary cycles in nontreated heifers (n, 63; mean, 20.8; SE, ± 0.4; CV, 13.9%). Treated heifers with short cycles (<15 days) had fewer CL (6.8 ± 1.4; mean ± S.E.) than heifers with intermediate cycles (15 to 27 days; 9.4 ± 0.6) or prolonged cycles (>27 days; 11.5 ± 0.7). Collection of an ovum from nontreated heifers resulted in shortening (P<.05) of the estrous cycle (n, 16; mean, 18.1 days) when compared to cycles from contemporary heifers in which collections were not done (n, 16; mean, 20.4 days).     

The fate of fertilized ova in rats receiving luteinizing hormone-releasing hormone (LRH).

The development and eventual fate of unimplanted fertilized ova in rats receiving pregnancy-inhibiting doses of luteinizing hormone-releasing hormone (LRH) were studied during the first 14 days of gestation. The results suggest that LRH accelerates passage of ova through the oviducts. Also, the zona pellucida of treated animals is retained by the majority of ova until day 7; zona-encased ova were observed as late as day 10. Elimination of the ova appears to occur by expulsion with uterine fluids through the vagina upon the animals' return to estrus.     

Plasma-Testosterone Development in Colony and Individually Housed Male Guinea Pigs

The behavioural development of male guinea pigs was studied in two colonies of 12 males and 12 females each, from their 30th to their 360th and 540th days of age respectively, living in enclosures of about 8 m². Changes in plasma-testosterone (T) concentrations were determined in individually (N = 8) (IM) and colony housed males (N = 10) (CM) from their 30th to their 360th days of age. In IM T-titers did not change significantly over time. In CM T-titers increased significantly from 30 to 90 and 120 days of age respectively, declined to lower levels until day 210, rose significantly at 240 days of age and remained on relatively high levels until day 360. CM showed significantly higher T-titers than IM on days 90, 120 and 240. In CM the first T-peak might reflect hormonal puberty. At this time CM, however, displayed low amounts of courtship and agonistic behaviour. The 2nd T-peak (240 days of age) occurred at an age when CM could achieve high ranking social positions for the first time which allowed reproductive success. It might be caused by the intense increase in sexual and agonistic behaviour during this period. In individual CM no general relationship between behavioural and endocrine development was found. At all ages T-titers poorly reflected behavioural differences between CM. Overall, androgen development did not follow a fixed temporal pattern during ontogeny but was strongly influenced by the social environment.     

Influence of ova within rat oviducts on spontaneous motility and on prostaglandin production

The present study was performed in order to explore the influence of ova present within rat oviducts on: a) tubal spontaneous motility and b) oviduct prostaglandin production. It was found that the isometric developed tension (IDT) of tubes isolated from proestrous rats (preovulatory oviducts) was significantly higher (P less than 0.01) than the IDT of tubes from rats at estrus and at metestrus (postovulatory oviducts). After flushing the oviducts with KRB solution (i.e., after removing existing ova) the IDT of the oviducts obtained from estrous rats increased significantly (P less than 0.01), whereas the IDT of tubes isolated from proestrous rats (i.e., preparations without ova) was not modified. On the other hand, isolated tubes containing their corresponding ova released into the suspending solution significantly more PGE1 than PGE2 or PGF2 alpha (P less than 0.005). It was particularly interesting to find that after flushing the oviducts, tissue production of PGE1, PGE2 and PGF2 alpha was similar. Finally, when dose response curves for PGE1 and for PGE2 on the spontaneous contractions of oviducts isolated from rats at proestrus, estrus and metestrus were constructed, both PGs evoked an inhibitory inotropic action. The ED50 for PGE1 in tubes from estrous rats was significantly smaller (P less than 0.01) than that for metestrous animals but significantly greater (P less than 0.01) than that observed in oviducts from proestrous rats. The ED50 for PGE2 did not change in the different tested periods of the sex cycle. Results reported herein suggest the possibility that the ova present within rat oviducts, may influence their own transport along the tubes by modifying the amount of prostaglandins produced by the oviducts or via their own prostaglandin synthesis.     

The effect of PMSG-priming on subsequent superovulatory response in dairy cows

Superovulation was induced in 56 dairy cows to evaluate the effect of two different regimens using pregnant mare serum gonadotropin (PMSG). Thirty-two cows (controls) were superovulated between Days 9 and 12 of the estrous cycle with a single dose of PMSG (2 800 IU), while remaining 24 cows (PMSG-primed) received 200 IU of PMSG on Day 4 of the estrous cycle and subsequently a single dose of PMSG (2 800 IU) between Days 8 and 12. The cows in both treatments were each given 0,5 mg of cloprostenol at 48 h after the superovulatory PMSG treatment. They were then artifically inseminated twice, 48 h and 72 h later. Embryos were recovered at sloughter between Days 2 and 5 of the cycle and morphologically evaluated. The number of corpora lutea (CL) in the ovaries of the cows was recorded. The mean number of CL (7.2 vs 17.8) was significantly higher (P 0.01) for PMSG-primed cows. The percentage of recovered ova (60.5 vs 70.2 %) and good embryos (79.3 vs 70.7%) were not significantly different between groups. The percentage of fertilized ova (91.4 vs 83.8%) was significantly (P 0.025) greater for the controls. Results of the study indicate that PMSG-priming increased the ovulation rate in the cows superovulated with PMSG.     

Estrous cycle stage-independent treatment of PMSG and hCG can induce superovulation in adult Wistar-Imamichi rats.

The estrous cycle influence on the number of ovulated eggs after injection of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) was investigated in 12, 18, and 24 weeks old adult female Wistar-Imamichi (WI) rats. PMSG (150 IU/kg) was injected at metestrus, diestrus, proestrus, or estrus, followed by hCG (75 IU/kg) 55 h later. Ovulation was induced at all ages and stages of the estrous cycle. The number of ovulated eggs was not affected by stage for similarly aged rats, however, the number of ovulated eggs obtained after treatment decreased with age. These results demonstrate that the PMSG/hCG treatment can induce ovulation at any stage of estrous cycle in WI rats and efficient superovulation at 12 weeks of age.     

Ovum recovery in superovulated cows and cleavage rates in the fertilized ova

Four groups of 10 cows were given one, two, three, or four thousand i.u. of PMS on Day 16 of the estrous cycle. On Day 19, the cows were injected with 10–15 mg of estrogen. At the ensuing estrus, the cows were bred and given 2,000 i.u. of HCG. The cows were slaughtered for ovum recovery 3–7 days after breeding. On the average, the cows produced 10.3 ± 1.6 ovulations with a range from 0 to 41. Each 1,000 i.u. of PMS was associated with an increase of 5.83 ± 1.18 in number of ovulations. The variation among cows within treatment groups was not attributable to differences in age or body weight. On the average, 51.5% of the ovulated ova were recovered, with the recovery rate being higher (64.2%) for ova in the oviducts and lower (34.2%) for those in the uterus. Ovarian length at the time of slaughter was directly related to PMS dosage and a smaller proportion of the ova were recovered from animals having largest ovaries. Only 38.4% of 211 recovered ova were fertilized. It was estimated that cleavages occurred at intervals of 12.6 ± 23.6 hours.     

Transvaginal ultrasound guided follicular aspiration of bovine oocytes

A transvaginal ultrasound guided follicular aspiration technique was developed for the repeated collection of bovine oocytes from natural cycling cows. In addition, the feasibility of using this method for collecting immature oocytes for in vitro embryo production was also evaluated. Puncturing of visible follicles for ovum pick-up was performed in 21 cows over a three month period. All visible follicles larger than 3 mm were punctured and aspirated three times during the estrous cycle on Day 3 or 4, Day 9 or 10 and Day 15 or 16. The mean (+/- SEM) estrous cycle length after repeated follicle puncture was 22.2 +/- 0.3 days. The mean total number of punctured follicles per estrous cycle was 12.6 +/- 0.3. The largest (P<0.05) number of follicles punctured (5.1 +/- 0.3) for ovum pick-up was on Day 3 or 4 of the estrous cycle. The overall recovery rate of 541 punctured follicles was 55%. Most oocytes (P<0.05) were aspirated from follicles smaller than 10 mm. Following in vitro maturation and fertilization (IVM/IVF), 104 oocytes were transferred to sheep oviducts. Six days later, 75 ova/embryos were recovered, after flushing the oviduct of the sheep, of which 24% developed into transferable morulae and blastocysts. In this study, a reliable nonsurgical, follicular aspiration procedure was used for the repeated collection of immature oocytes which could be used successfully for in vitro production of embryos. This procedure offers a competitive alternative to conventional superovulation/embryo collection procedures.     

Pregnancy resulting from cattle oocytes matured and fertilized in vitro

Follicular oocytes (n = 81) collected from cattle at a local slaughterhouse were matured and fertilized in vitro. Of 27 ova 19 (70%) were penetrated by spermatozoa and 40/54 (74%) inseminated ova transferred surgically to the oviducts of a synchronized heifer were recovered by non-surgical flushing of the uterine horns 6 days later. Of the 40 ova 15 (38%) were at the morula, early blastocyst or diminutive morula stages. Culture in vitro sustained further development of all embryos and 9 were expanding or expanded blastocysts. One pregnancy resulted from non-surgical transfer of 2 blastocysts. The results demonstrate that immature oocytes from cattle can be matured and fertilized in vitro, subsequently develop to the blastocyst stage, and develop into a normal pregnancy after non-surgical transfer.     

Superovulatory response of river bufalo (Bubalus bubalis )

The ovarian response of 25 buffalo-cows was visually assessed, and their oviducts and uteri separately flushed 3 to 6 d post superovulatory estrus at slaughter. Ten buffalo-cows slaughtered on Days 5 and 6 were examined per rectum for corpora lutea (CL) and follicles > 8 mm prior to slaughter, and the estimate was compared later with the actual ovarian response. Five out of the ten buffalo-cows were nonsurgically flushed in vivo on Day 5 of the estrous cycle, a day before slaughtering, and as a result, six ova/embryos were recovered. After the flushing of the reproductive tract at slaughter, one more ovum was recovered from the uterus of each of the three buffalo-cows. As a result of treatment of three groups of five buffalo with 3000 IU pregnant mare serum gonadotrophin (PMSG) on Days 6, 10 or 14 of the estrous cycle, 3.8, 6.2 and 3.4 CL on the average were recovered, respectively (Experiment I). A mean number of 8.8 and 9.0 CL, respectively, was obtained in two groups of five buffalo each, after treatment with 40 mg of follicle stimulating hormone (FSH) on Day 10 of the stage of the estrous cycle (Experiment II) and 3000 IU PMSG regardless of the stage of cycle (Experiment III). The percentage of ova/embryos recovered in the three experiments was 32.8, 20.4 and 22.2, respectively.     

Inhibition of sperm transport and fertilization in superovulating ewes

In Experiment 1, all ewes were treated with follicle stimulating hormone (FSH-P) to induce superovulation. Ewes came into natural estrus or were treated with prostaglandin F(2)alpha (PGF(2)alpha) or 6-methyl-17-acetoxyprogesterone (MAP) to regulate the time of estrus. The ewes were mated during estrus and necropsied 3 h after mating. Regulation of estrus with either compound reduced the number of sperm recovered from the cervix, uterus, and oviducts and increased the proportions of sperm recovered from the cervix and uterine body that were immotile, dead, or had disrupted membranes. In Experiment 2, all ewes were in natural estrus. They either ovulated naturally or were superovulated, and ewes in each group were necropsied at 3 or 23 h after mating. Superovulation reduced the number of sperm in oviducts, uterus, and anterior segments of the cervix at both time intervals and increased the proportions of sperm that were immotile, dead, or had disrupted membranes. In Experiment 3, of 3x2 design, ewes were in either natural estrus or estrus regulated with PGF(2)alpha or with MAP; they ovulated naturally or were superovulated. Ewes were necropsied 3 d after mating and ova were examined. Both regulation of estrus and superovulation reduced the proportion of ova that were fertilized and reduced the number of accessory sperm attached to fertilized ova.     

Aging of mouse eggs in vivo and in vitro

Morphological and cytochemical (acid phosphatase) changes associated with mouse ova and cumulus cells aged within the oviducts (in vivo) or in culture (in vitro; 1–24 hours postovulation) have been investigated. Structural alterations of cumulus cells were apparent immediately after ovulation and included nuclear pycnosis and cytoplasmic vacuolization. Nevertheless, approximately 30% of the cumulus masses examined contained cells that plated out when cultured and remained viable for up t o three days in vitro. From 12 t o 24 hours postovulation almost all cumulus cells of specimens aged in vivo showed signs of degeneration. Disruption of the meiotic spindle and an increase in acid phosphatase positive organelles were characteristic of in vivo and in vitro aging ova. The percentage of fragmented eggs obtained from super-ovulated (5 IU PMS followed by 5 IU HCG) mice approximately one and 24 hours postovulation was not significantly different. Eggs obtained from superovulated animals and aged in vitro for 24 hours yielded significantly more fragmented ova. Fragmented eggs were not obtained from cycling females on the morning of estrus. When such eggs were cultured in vitro for 24 hours the percent fragmentation was significantly lower than that for aged eggs obtained from super-ovulated mice. These results indicate that 1) similar morphological alterations occur among cumulus cells and eggs aged either in vitro or in vivo, 2) ova from superovulated mice do not constitute a homogeneous population and 3) the method of superovulation employed in this study induces the ovulation of a relatively large group of eggs that are susceptible to fragmentation when cultured in vitro.     

Changes in germ cell population in young and adult female mice from two inbred strains: CBA/Kw and KE

Female mice from two inbred strains CBA/Kw and KE differ markedly in fertility. The gametes of females from KE strain are of poorer quality than those of CBA/Kw. We analyzed the number of oocytes per ovary in KE and CBA/Kw mice aged 5, 25, 90, 180 and 360 days. The ovaries were dissected and processed according to the routine histological methods. In case of five-day-old females we used a modified distributed point counting method while in order to examine the gonads of older females, the nucleoli counting method was applied. In general, we observed gradual decrease in germ cell number throughout the whole life of females from both strains. The noticeable wave of oocyte loss occurs between 5th and 25th days of life. The mice from KE inbred strain on day 25th (1650 +/- 322 vs. 1140 +/- 210) and 90(th) (1040 +/- 211 vs. 692 +/- 89) days have significantly (p<0.005) more germ cells than the females from CBA/Kw strain. In older females the differences were not statistically significant. Interestingly, CBA/Kw females were found to have more rapid loss of primordial follicles throughout their lives. This can explain their shortened reproductive lifespan which was observed earlier.     

Superovulatory responses of Holstein cows

Approximately 1000 registered cows and heifers were superovulated one to 10 times. Nonsurgical embryo recoveries were performed on all donors which exhibited estrus. Healthy donors produced more total ova and cleaving embryos and had a higher ovum recovery rate, fertilization rate and pregnancy rate from embryos transferred than did cows classified as infertile. While ovum number was not affected during 10 repeated superovulations, fertilization rate and embryo number decreased. The number of ova recovered from healthy cows was affected by season, and from infertile cows by the day of the estrous cycle on which FSH was started and by the number of days since calving. More ova were recovered from infertile cows synchronized with prostaglandins prior to superovulation than following a natural estrous cycle. The number of embryos recovered from infertile cows was affected by age and from healthy cows by daily milk production. Fertilization rates in both healthy and infertile cows were affected by age, time since calving, daily milk production, day of cycle FSH was injected and season. There was no effect of the day of recovery on the number of ova or embryos recovered from healthy or infertile cows.     

Role of decreased numbers of follicles on reproductive performance in young and aged rats

The primary objectives of this study were to: 1) determine if removal of 1.5 ovaries from young rats would mimic reproductive characteristics that normally occur with advancing age and 2) determine if removal of 1.5 ovaries from aged rats would further advance the process of reproductive aging. Removal of 1.5 ovaries increased the number of young (P less than 0.05) and old (P less than 0.01) rats that exhibited abnormal estrous cycles. In addition, concentrations of follicle-stimulating hormone (FSH) were higher at both ages in the groups with half an ovary. The increased concentrations of FSH are consistent with a decrease in the number of growing follicles after removal of 1.5 ovaries. All groups had lower concentrations of estradiol (E2) than young controls. There was a significant increase in the number of abnormal embryos with age and removal of 1.5 ovaries when rats were mated during a 5-day estrous cycle, but there was no effect if they were mated during a 4-day estrous cycle. From the results of this study, we conclude that the reduction in ovarian tissue in young and aged rats mimicked several reproductive characteristics of advancing age. Also, an effect of aging on the hypothalamus was evident in this study.     

Factors affecting success of embryo collection and transfer in large dairy herds

Our objective was to evaluate factors that affected the success of embryo transfer programs in large dairy herds. Non-lactating donor cows produced a larger number of ova/embryos (P<0.01) and viable embryos (P<0.01) than lactating cows. The interaction between season and donor class was correlated with the proportion of ova/embryos classified as fertilized (P=0.03), because lactating donors had fewer fertilized ova in the summer. There was no correlation between 305-day mature equivalent milk yield and response to superstimulation. Although the interval between superstimulation protocols was correlated with the number of ova/embryos (P=0.03), there was no correlation with the number of viable embryos. Pregnancy per embryo transfer (P/ET) in heifer recipients was correlated with embryo quality grade (P<0.01), season (P=0.04), and whether embryos were fresh or frozen/thawed (P<0.01). Lactating recipient cows tended to have a lower rate of P/ET during the summer (P=0.12 to P=0.08). Synchronization protocols tended to be (P=0.06; Herd 1) or were (P=0.02; Herd 2) correlated with P/ET. Lactating cows receiving vitrified IVF embryos had a lower (P=0.01) P/ET than those receiving fresh IVF embryos, especially in the summer (P=0.09). Milk yield was not correlated with P/ET. The use of heat abatement systems is critical to improve embryo production and P/ET. Synchronization protocols that optimized synchrony of ovulation may increase fertility of recipient cows and eliminate the need for estrous detection.