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Observations on the use of human chorionic gonadotrophin (HCG) during the post-insemination period on conception rates in synchronized beef cows with sub-optimum reproductive performances.

Oestrus was synchronized in 46 Afrikaner and Mashona beef cows by two injections of cloprostenol 11 days apart. All cows had a history of sub-optimum reproductive performance. Cows were inseminated up to three times after the second cloprostenol injection on the basis of observed oestrus and changes in the conductivity of cervical mucus. Half the cows received daily injections of 1000 i.u. HcG from days four to 19 after their last insemination; the other half received daily injections of 2 ml saline over the same period. Concentrations of progesterone in plasma was determined from samples taken on days 6, 8 and 22 after the last insemination. Treatment did not significantly affect conception rate and overall conception rate was 39 per cent. On day 8 after insemination none of the 8 cows that had progesterone levels of less than 1 ng/ml were pregnant when examined at day 70. Mean progesterone concentrations were not significantly different between treated and control cows on days 6 and 8, but were significantly higher (P<0,05) in treated cows by day 22. The practical significance of using HcG to stimulate luteal function in the early post-inseminaion period is discussed.

Journal Article↗

The integration of the transfer of frozen embryos with artificial insemination in a commercial beef herd.

This trial was designed to examine the integration of the transfer of frozen embryos and artificial insemination (A.I.) in a commercial herd of beef cows. Embryos were collected nonsurgically from 15 superovulated cows and all embryos judged viable (n = 69) were frozen in 0.25 ml plastic A.I. straws. Glycerol was used as a cryoprotectant and the embryos were frozen with omission of the traditional "seeding" step. On three consecutive days immediately preceding the onset of a 60-day breeding season, 54 embryos were transferred to multiparous cows with calves at foot. The remaining 82 cows in the same herd were used as controls. Cows receiving embryos were maintained under observation, but not inseminated for 21 days, after which cows exhibiting signs of estrus were inseminated up to Day 60. Cows in the control group were inseminated at observed estrus throughout the 60-day period. The overall pregnancy rate for the experimental and control groups did not differ (77.8 % and 73.2 % respectively). Of the experimental cows, 24.1 % conceived to the embryo transfer, and 53.7 % to A.I. The mean calving to conception interval for the experimental cows was 96.3 days (median 101 days) and for the control cows it was 92.3 days (median 88 days). The calving pattern in the experimental cows was biphasic, calvings resulting from embryo transfer of A.I. accounting for the two peaks. Reconception rates in the following breeding season were not different between the embryo transfer or A.I. groups or between experimental and control herds. Weaning masses of calves born of embryo transfer were significantly higher than the other groups, but calves resulting from A.I. in the experimental herd were not different from calves in the control herd. It was concluded that the transfer of frozen embryos could be successfully integrated into an A.I. program with a limited breeding season without detrimentally affecting the overall reproductive performance of the recipient herd.

Journal Article↗

Accuracy of predicting the LH surge and optimal insemination time in Holstein heifers using a vaginal resistance probe.

Monitoring changes in vaginal electrical resistance in association with ovarian follicular development during proestrus may provide an alternative to visual estrus detection of cattle for properly timing insemination. In two experiments 94 Holstein heifers were synchronized into estrus with 2 injections of prostaglandin F(2) alpha (PG) 11 d apart. Beginning 12 h prior to the second PG treatment, blood samples and vaginal resistance measurements were taken every 12 h for 60 h, and then every 6 h for the following 48 h. In both Experiments I and II, the lowest resistance value was found to be highly correlated with the luteinizing hormone (LH) preovulatory surge (r = 0.67 P<0.001). The linear regression of time from PG to the LH surge (Y) on time from PG to the lowest resistance value (x) was defined by Y = 17.49 + 0.71 (x) (R(2) = 0.45). This suggests that the lowest resistance value can predict the LH surge and, therefore, the appropriate time to inseminate heifers relative to the expected time of ovulation. This concept was tested in Experiment II by assigning 50 heifers to be inseminated after PG treatment on the basis of their vaginal resistance pattern, by appointment 66 h after PG, or by visual observation of standing estrus. Conception rates were 58, 50 and 50% in these groups respectively, and did not differ significantly. Overall, these results suggest that measurement of vaginal resistance patterns reliably predict the LH surge and can be successfully utilized to inseminate cattle.

Journal Article↗

Single appointment insemination for heifers after prostaglandin or progestin synchronization of estrus.

Pregnancy rates to a single appointment insemination were compared in seven groups of beef or dairy heifers following estrus synchronization with the prostaglandin F(2a) product, cloprostenol, versus the progestín product, Syncro-Mate-B. For cloprostenol synchronization, two injections of cloprostenol were given 11 d apart, with insemination occurring at 61+/-1 h after the second injection. The Syncro-Mate-B treatment consisted of a norgestomet/estradiol injection and a norgestomet implant on Day 0, followed by insemination at 49+/-1 h after implant removal on Day 9. Treatments were coordinated so all heifers in each group were housed together at the time of and for 48 h before a common insemination time so that the treatment received by individual heifers was not known. Overall pregnancy rates did not differ between treatment groups: 42% for 111 heifers treated with cloprostenol and 38% for 108 heifers treated with Syncro-Mate-B. Four blood samples per heifer taken during each replicate showed that two groups (n = 67) had many noncyclic heifers, while five groups (n = 152) had very few. Differences in pregnancy rates between these categories existed for both estrus synchronization methods, 18 versus 53% for cloprostenol and 21 versus 45% for Syncro-Mate-B. Thus the two methods were equally effective for cyclic heifers and equally ineffective for noncyclic heifers.

Journal Article↗

Time of ovulation in the South Australian Merino ewe following synchronization of estrus. 2. Efficacy of GnRH treatment and its relevance to insemination programs utilizing frozen-thawed semen.

In a study of the time of ovulation following synchronization of estrus in the ewe, the effect of time of treatment with GnRH (24 vs 36 h after pessary removal) and dosage (6.25 to 100 ug per ewe) were examined. All treatments synchronized the time of ovulation irrespective of when untreated ewes commenced to ovulate. As part of an evaluation of GnRH treatment in artificial insemination programs, an assessment was made of the quality of eggs obtained from control ewes and ewes treated with GnRH at either 24 or 36 h after pessary removal. Treatment at 24 h increased the number of retarded embryos (P < 0.01) and unfertilized ova (P < 0.01) collected per ewe, reduced the number of embryos collected per ewe (P < 0.01), and reduced (P < 0.05) the percentage of pregnant ewes compared with other groups. However, there were no differences between control ewes and ewes treated with GnRH at 36 h. GnRH treatment at 36 h was consequently examined as a means of improving conception rates following the intrauterine insemination of frozen-thawed semen. Insemination of GnRH-treated ewes 8 to 12 h before the median time of ovulation resulted in a nonsignificant increase (range 5.7 to 7.3%) in the percentage of ewes of mature age which became pregnant. Insemination 0 to 4 h before the median time of ovulation resulted in a nonsignificant decrease in the percentage of pregnant ewes. GnRH treatment did not influence the number of fetuses per ewe. Reasons for the failure of this treatment to significantly improve ewe fertility are discussed.

Journal Article↗

A technique for transcervical intrauterine insemination of ewes.

In commercial artificial insemination (AI) of sheep, fresh extended semen is deposited into the vagina or cervical os, or fresh extended or frozen semen is placed laparoscopically into the uterus. Transcervical intrauterine insemination of the ewe is not used commercially. In this study, methods of restraint and instrumentation for AI were evaluated and modified to produce a transcervical intrauterine technique suitable for commercial application. Four methods of restraint, four vaginal specula, three forceps and four instruments suitable for transcervical passage were compared. From these comparisons a technique was developed in which the ewes were positioned in dorsal recumbency with their hindquarters elevated. The vagina was dilated using a duck-billed speculum, the cervix was grasped and retracted using forceps, and an inseminating instrument was introduced into the cervical opening and manipulated through the cervical canal. The technique was repeated on 89 mature, multiparous ewes: the difficulty in locating the cervical opening, the force required to retract the cervix and the time required to penetrate into the uterus were recorded. Uterine penetration was achieved in 82% of the ewes. This technique has the potential to be applied in commercial artificial insemination programs of sheep.

Journal Article↗

Effect of side of insemination on transuterine transport of spermatozoa in superovulated dairy cattle.

Eleven superovulating Friesian lactating cows were inseminated deep into one uterine horn with one unit of frozen semen, containing 2.5 million total spermatozoa, with more than 40% postthaw progressive motility and with 14% morphologically abnormal spermatozoa. Semen was deposited into the right or left uterine horns of alternate cows. There was no difference in the proportions of fertilized recovered ova from ipsilateral horns between right and left inseminations (P>0.05). The fertilization rate in the contralateral horns was higher (P<0.01) for right uterine horn insemination (50%) than for left uterine horn insemination (15.6%).

Journal Article↗

Importance of cumulus cells and insemination intervals for development of bovine oocytes into morulae and blastocysts in vitro.

Experiments were carried out to investigate influences of cumulus cells and insemination intervals on bovine in vitro fertilization (IVF) and early development. Cumulus-encased oocytes, aspirated from 2- to 5-mm ovarian follicles at slaughter, were incubated 24 hours for maturation in the presence of 20% proestrous (Day 20) cow serum and 100 microg LH/ml. Ova with mature (expanded) cumuli oophori were inseminated and removed from sperm-containing droplets (50 microl) after 6, 12, 24 or 48 hours. After maturation, some ova were removed from their surrounding cumulus cells and otherwise treated in the same way. The highest proportion of ova that cleaved (50/57, 87.7%) resulted from the 24-hour insemination group; this was significantly higher (P<0.05) than for 6-hour (35.1%) and 12-hour group (49.3%), but not for the 48-hour group (73.0%). A significantly (P<0.05) higher proportion of cleaved ova developed into morulae/blastocysts (28%) from the 24-hour group. Removal of cumulus cells before IVF resulted in lower cleavage rates; the morula/blastocyst stage was reached only when the denuded ova were with sperm for 48 hours. In additional experiments, cumulus cells recovered from follicular aspirates were cultured in HEPES-M 199 with 10% Day-20 serum, and 0, 10 and 100 microg LH/ml and resulting monolayers were used for zygote culture. The developmental stages reached after IVF were not altered by LH treatment of supporting cumulus cells. A 24-hour insemination interval with subsequent culture on a cumulus cell monolayer resulted in optimal in vitro development.

Journal Article↗

The initial fertilizing capacity of longerm-stored liquid boar semen following pre- and postovulatory insemination.

In pigs, high variation is seen in the duration of estrus and in the time of ovulation. This is one of a wide range of factors not related to semen quality, which possibly influences the results of field insemination trials. Experiment 1 (n=81 gilts) was performed to determine the influence of the time of ovulation on the fertilizing capacity of liquid boar semen stored up to 118 h. The objective of Experiment 2 (n=102 gilts) was to study the fertilizing potential of semen stored up to 120 h in 2 different extenders, Androhep and Beltsville Thawing Solution (BTS), by means of postovulatory AI. Inseminations were performed 0 to 4 h after ovulation in order to standardize the trial conditions. Fertilization rates based on Day-2 to Day-4 embryos, and the number of accessory spermatozoa per zona pellucida did not differ between semen stored for 0 to 48 and 48 to 87 h in gilts ovulating within 12 after insemination (Experiment 1). Gilts with an interval of 12 to 24 h between AI and ovulation had lower fertility results using semen stored for more than 48 h. A further decrease was observed when semen storage exceeded 87 h in those gilts ovulating later than 24 h after insemination. The time of ovulation has to be considered as being a major factor of variation in the fertility results of AI trials. In Experiment 2, fertilization rates and numbers of accessory spermatozoa decreased between semen stored for 0 to 24 and 24 to 48 h in BTS, and between semen stored for 0 to 24 and 48 to 72 h in Androhep. Significant differences in fertility between diluents were seen only when using semen stored for more than 96 h, with semen extended with Androhep giving the higher results. The results indicate that the decrease in fertilizing capacity due to in vitro aging of spermatozoa cannot be prevented even during the first days of storage.

Journal Article↗

In vitro fertilization-intracytoplasmic sperm injection split: an insemination method to prevent fertilization failure.

OBJECTIVE: To determine whether the performance of intracytoplasmic sperm injection (ICSI) in cases of unexplained infertility and of borderline semen parameters can prevent fertilization failure. DESIGN: Prospective cohort study. SETTING: Hospital-based assisted reproductive technology program. PATIENT(S): Sixty couples with unexplained infertility (group 1) and 50 couples with borderline semen (group 2). INTERVENTION(S): Conventional IVF and ICSI were performed on sibling oocyte cumulus complexes. MAIN OUTCOME MEASURE(S): Rate of fertilization according to patient diagnosis. RESULT(S): The fertilization rate per oocyte cumulus complex in couples with unexplained infertility (group 1) was significantly higher with ICSI (65.3%) compared with IVF insemination (48.1%). Fertilization rates per OCC in couples with borderline semen (group 2) were not significantly different between ICSI (58.1%) and IVF (56.7%). In group 1, failure of fertilization after IVF insemination occurred in 10 of the 60 couples (16.7%) and in none with ICSI. In group 2, 2 of 50 couples (4.0%) failed fertilization by IVF, and 1 (2.0%) failed with ICSI. No couple in group 2 failed both IVF insemination and ICSI. CONCLUSION(S): In couples with unexplained infertility, a higher fertilization rate was achieved through ICSI compared with conventional IVF. No such benefit could be demonstrated for couples with borderline semen parameters. The use of ICSI rescued 12 of 110 cycles (10.9%) where IVF failed. Adoption of the ICSI-IVF insemination split in cases of unexplained infertility may help eliminate fertilization failures.

Cohort Studies↗

Effect of the number of inseminating sperm and the follicular stimulation protocol on in vitro fertilization of human oocytes in male factor and non-male factor couples.

The effect of sperm concentration and follicular stimulation protocol on in vitro fertilization of human oocytes is not well established. Comparison was made of three inseminating concentrations (250,000, 375,000, and 500,000 progressively motile sperm/oocyte) and three protocols (human menopausal gonadotropin [hMG], clomiphene citrate [CC], and combination hMG/CC) on the fertilization rate of mature and immature oocytes in couples with male factor and non-male factor infertility. In non-male factor couples, total fertilization rates for CC, hMG, and hMG/CC were 70.3%, 54.5%, and 68.8%, respectively, while total fertilization rates at the varying number of inseminating sperm were not significantly different. Mature oocytes were more likely than immature oocytes to fertilize. Among semen male factor couples, there was no difference in fertilization by stimulation protocol; however, insemination with the higher number of inseminating sperm resulted in an increased fertilization rate.

Clomiphene↗

Fertilization of human oocytes in relation to varying delay before insemination.

Fertilization and pregnancy rates in an in vitro fertilization and embryo transfer program were studied after a range of insemination times of between 1 and 26 hours after oocyte recovery. There was no significant variation in fertilization rate across this range. The pregnancy rate showed no significant variation for insemination between 3 and 16 hours after aspiration. However, it was disappointing at 2 hours (3%), and no pregnancies were achieved from the nine patients whose ova were inseminated 20 or more hours after aspiration. It is concluded that mature oocytes can be inseminated in vitro at any time between 3 and 16 hours after aspiration and still retain the same potential to produce a pregnancy.

Female↗

Increase of pregnancy rate in dairy cattle after preovulatory follicle palpation and deep cornual insemination.

A total of 334 first-service lactating cows in natural estrus were used in the study. Semen was deposited into the uterine body of 174 cows and deep into the uterine horn ipsilateral to the side of impending ovulation of 160 cows. In both groups, insemination was performed within the interval of 50 to 100 d postpartum at 8 to 15 h after estrus detection and after preovulatory follicle palpation. Pregnancy rates were determined by palpation per rectum 50 d post insemination. The pregnancy rate was higher (P < 0.05) for deep uterine horn insemination (70.62%) than for uterine body insemination (60.34%).

Journal Article↗

Sperm pre-incubation prior to insemination affects the sex ratio of bovine embryos produced in vitro.

The objective of the present study was to determine whether sperm incubation prior to oocyte insemination in vitro affects the sex ratio of resulting blastocyst. Cumulus-oocyte-complexes (COCs) collected from slaughterhouse ovaries were matured in vitro and inseminated with frozen-thawed semen of three proven artificial insemination (AI) bulls pre-incubated in vitro in Sperm-Talp for 6 and 24 h. On day-9 blastocysts were collected and processed for sex determination. More than 80% of blastocyst were successfully sexed. There were no significant differences in cleavage and blastocyst rates using sperm pre-incubated for 6 h as compared with the 0-h pre-incubation control group. The cleavage and blastocyst rates were significantly lower in the 24-h pre-incubation group. The male to female ratio, when compared with the theoretical 1 : 1, differed significantly in favour of females among hatched (viable) blastocysts derived from sperm pre-incubated for 24 h prior to insemination as well as among all blastocytsts in the 6-h group. Moreover, when the sperm treatment was considered, the sex ratio was affected only among hatched blastocysts in 24-h pre-incubation group. It was concluded that prolonged sperm pre-incubation influences the rate of development and the sex ratio among hatched blastocysts.

Animals↗

Reduced polyspermic penetration in porcine oocytes inseminated in a new in vitro fertilization (IVF) system: straw IVF.

High incidence of polyspermy is still a major problem in the in vitro fertilization (IVF) of porcine oocytes matured in vitro. This study was designed to examine whether embryo cryopreservation straws can be used to conduct IVF in porcine oocytes. The efficiency of this system was further compared with traditional microdrop IVF. Immature oocytes were aspirated from antral follicles and matured in vitro. After maturation, oocytes were inseminated either in straws or in microdrops with frozen-thawed boar spermatozoa. For straw IVF, sperm concentration and the presence of air columns between insemination segment and oil column were examined. Sperm-oocyte binding and cortical granules (CGs) before and after sperm penetration were examined by confocal microscopy. When various sperm concentrations were used for IVF in the straws with air columns, it was found that 5 x 106 cells/ml of sperm concentration was the optimal concentration; a high penetration rate (94.0%) and normal fertilization (oocytes with both male and female pronuclei) rate (38.2%) were obtained. Increasing sperm concentration to 10 x 106 cells/ml increased polyspermic penetration (61.9%) without affecting sperm penetration (86.9%). Reducing sperm concentration to 1 x 106 cells/ml reduced polyspermic penetration (25.6%), but sperm penetration rate (69.9%) was also reduced. When IVF was conducted in the straws with or without air columns, and in the microdrops, it was found that sperm penetration in the straws with air columns (96.5%) was significantly (p < 0.05) higher than that in the straws without air columns (81.7%) and in the microdrop (72.9%). However, the incidence of polyspermic penetration in the straws with air columns (34.2%) and without air columns (36.6%) was significantly (p < 0.05) lower than that (52.4%) in the microdrops. The number of spermatozoa bound to the oocytes was increased gradually in the straws but not in the microdrops in which more spermatozoa bound to the oocytes soon after insemination. CG exocytosis was more complete and faster in the oocytes inseminated in the straws than in the microdrops. These findings indicate that IVF of porcine oocytes in the straws provides a better condition in which more oocytes are fertilized normally than that in the microdrop IVF.

Animals↗

Reproductive physiology and development of artificial insemination technology in killer whales (Orcinus orca).

Research was conducted to define the basic reproductive physiology of killer whales (Orcinus orca) and to use this knowledge to facilitate the development of artificial insemination procedures. The specific objectives were 1) to determine the excretory dynamics of urinary LH and ovarian steroid metabolites during the estrous cycle; 2) to evaluate the effect of an exogenously administered, synthetic progesterone analog on reproductive hormone excretion; 3) to validate the use of transabdominal ultrasound for ovarian evaluation and timing of ovulation; 4) to examine the quality of semen after liquid storage and cryopreservation; and 5) to develop an intrauterine insemination technique. Based on urinary endocrine monitoring of 41 follicular phases and 26 complete cycles from five females, estrous cycles were 41 days long and comprised a 17-day follicular phase and a 21-day luteal phase. A consistent temporal relationship was observed between peak estrogen conjugates and the LH surge, the latter of which occurred approximately 0.5 days later. Two animals placed on oral altrenogest (three separate occasions for 30, 17, and 31 days, respectively) excreted peak urinary estrogen concentrations 25 days after withdrawal that were followed by sustained elevations in urinary pregnanediol-3alpha-glucuronide excretion. Mean preovulatory follicle diameter was 3.9 cm (n = 6), and ovulation occurred 38 h (n = 5) after the peak of the LH surge. Based on visual estimates of motility, liquid-stored semen maintained 92% of its raw ejaculate sperm motility index (total progressive motility x kinetic rating [0-5 scale, where 0 = no movement and 5 = rapid progressive movement]) when held at 4 degrees C for 3 days postcollection. Semen cryopreserved using a medium freezing rate demonstrated good postthaw total motility (50%), progressive motility (94%), and kinetic rating (3.5). Insemination during eight estrous cycles resulted in three pregnancies (38%), two from liquid-stored and one from cryopreserved semen. Two calves were delivered after gestation lengths of 552 and 554 days, respectively. These data demonstrate the potential of noninvasive endocrine monitoring combined with serial ultrasonography to improve our understanding of the reproductive biology of cetaceans. This fundamental knowledge was essential for ensuring the first successful conceptions, resulting in live offspring, using artificial insemination in any cetacean species.

Acrosome↗

Relationship between longevity of spermatozoa after insemination and the percentage of normal embryos in brown marsupial mice (Antechinus stuartii).

Twenty-six female brown marsupial mice in a laboratory colony were mated at intervals ranging from 1 to 20 days between coitus and ovulation. The numbers of corpora lutea and normal embryos were counted. A multiple regression model examined the parabolic relationship between the proportion of normal embryos and the time from coitus to ovulation. The proportion of normal embryos increased until a mean of 9.5 days and decreased thereafter. This relationship was independent of the year of breeding and the number of corpora lutea. After survival of spermatozoa for up to 13 days in the female reproductive tract, the fertility levels of females was 88-92%. Low fertility levels after 13 days appeared to be due to a decrease in the number of spermatozoa. Reproductive tracts from 7 females killed after insemination and examined histologically showed many spermatozoa in the isthmus of the oviduct and the uterus at 5 days post coitum; spermatozoa confined to the isthmus between 6 and 13 days; and few spermatozoa in the isthmus at 14 days after copulation. A comparison between the fertility levels in the females which had been inseminated once and a further 17 females which had been inseminated 2 or 3 times suggested that spermatozoa from 2nd and 3rd inseminations can contribute spermatozoa for fertilization. In these females fertility levels did not decline with time after the first mating.

Animals↗

Pregnancy after subzonal insemination with spermatozoa lacking outer dynein arms.

The absence of outer dynein arms in the sperm flagellum induces an abnormal movement pattern associated with male infertility. These spermatozoa can decondense in zona-free hamster oocytes but result in a very low fertilization rate in in vitro fertilization. We hypothesized that subzonal insemination could help achieve fertilization and pregnancy. A randomized prospective trial (five couples, five cycles) comparing subzonal insemination (n = 31 oocytes) and routine IVF (n = 23 oocytes) was carried out. Oocytes were microinjected with 8.5 +/- 3.6 spermatozoa. In a second series (nine cycles), all the oocytes were microinjected with 10.5 +/- 4.3 spermatozoa. In the randomized series, the fertilization rate was 16.1% without polyploidy, whereas no fertilization was obtained after control IVF insemination. In the second series involving nine couples, six of whom were included in the first series, the fertilization rate increased to 57.8% with a 27.8% polyspermic rate. Eighty-eight per cent of the zygotes cleaved normally (29 out of 33). A total of 11 embryo transfers resulted in three pregnancies, one of which terminated one month later, a second being ongoing and the third delivering a healthy girl. A 21.4% pregnancy rate per cycle, with a 37.5% pregnancy rate per couple, justifies the use of subzonal insemination to treat this particular flagellar dyskinesia.

Adult↗