The use of synthetic gonadotropin releasing hormone treatment in the collection of sheep embryos

Gonadotropin releasing hormone (GnRH) treatment was examined as a means of improving the efficacy of embryo collection in the sheep following intrauterine insemination of frozen-thawed semen. In summary, treatment consistently improved fertilization rates and the number of fertilized ova collected per ewe was enhanced compared with untreated ewes. The yield of fertilized ova in ewes treated with follicle stimulating hormone (FSH) was maximized by administering GnRH 36 h after progestagen treatment; 24 h was the preferred time in ewes treated with pregnant mare serum gonadotropin (PMSG). There was a significant (P < 0.001) increase in the percentage of unfertilized ova in the former treatment when GnRH was given at 24 h. An examination of the time of insemination (0, 6, 12 and 18 h before the median time of ovulation) indicated that fertilization rates were highest when insemination occurred at 6 h in both GnRH-treated ewes and in untreated ewes. In GnRH-treated ewes, the recovery of ova was lowest when insemination occurred at the time of ovulation. The number of motile frozen-thawed spermatozoa required for fertilization following treatment was estimated to be approximately 20 x 10(6) per uterine horn. GnRH-treatment also improved the yield of fertilized ova in sheep that were naturally mated, although this yield was lower than that obtained with intrauterine insemination of frozen-thawed semen. It is concluded that fertilization failure, a major problem in sheep embryo collection, can be eliminated through judicious use of GnRH treatment and properly timed intrauterine insemination.     

Fertility of Deep Frozen Boar Spermatozoa at Various Intervals between Insemination and Induced Ovulation

This study was performed to investigate the influence of boars and thawing diluents on the fertilizing capacity of deep frozen spermatozoa at various intervals between inseminations and ovulation. Forty-four Swedish crossbred gilts were inseminated following injection of HCG late in the prooestrus. Inseminations were performed 22, 28, 34 and 38 hrs. after injection of HCG. Ovulation was expected to occur 40 hrs. after injection of HCG. Two boars, previously tested for fertility with frozen semen, supplied the spermatozoa. Roar seminal plasma and OLEP were utilized as thawing diluents. The gilts were slaughtered 32–48 hrs. after estimated ovulation. The genital tracts were removed immediately after stunning and bleeding and the numbers of recent ovulations, recovered ova and fertilized ova were recorded. Additionally recovered ova were classified according to estimated numbers of spermatozoa attached to the zona pellucida. Similar fertilization rates were obtained when inseminations were performed 2 and 6 hrs. before estimated ovulation. A clear decline in fertility appeared when inseminations were performed earlier than 6 hrs. before expected ovulation. The results were influenced by the boars as well as by the thawing diluents. Seminal plasma yielded a higher fertilization rate than OLEP in inseminations performed 2 hrs. before estimated ovulation. The boars yielded similar fertility in inseminations performed 2 hrs. before estimated ovulation. With increasing intervals between inseminations and ovulation the difference between the boars increased. The single gilt in which fertilized ova were found after insemination 18 hrs. before ovulation was inseminated with spermatozoa from the superior boar, thawed in seminal plasma. The present results indicate that spermatozoa with low resistance to freezing-thawing have a short fertile life in the female genital tract after insemination.     

The effects of spermatozoal irradiation with X-rays on chromosome abnormalities and on development of mouse zygotes after delayed fertilization

The chromosome complements of zygotes derived from oocytes aged post ovulation and fertilized in vivo with X-ray-irradiated sperm were studied. Ovulation was induced by an injection of luteinizing hormone-releasing hormone (LHRH) at pro-estrus and fertilization was achieved by artificial insemination at 13 h and 24 h after LHRH in order to obtain embryos from unaged and aged (12 h post-ovulation) oocytes respectively. Post-ovulatory aging prior to fertilization did not significantly affect the percentage of zygotes with irradiation-induced chromosome abnormalities. However, post-ovulatory aging had a negative effect on the morphology of male as well as female pronuclear chromosomes of the first cleavage metaphase. When fertilized with control spermatozoa this effect was apparent in both the male and the female pronucleus. When unaged oocytes were fertilized with X-irradiated spermatozoa chromosome morphology was also adversely affected in both pronuclei. In zygotes from aged oocytes, there was an extra negative effect of X-rays on the male pronuclear chromosomes only. After fertilization with X-irradiated sperm 27% of zygotes from aged oocytes were arrested at interphase compared to 7% from unaged oocytes. We suggest that post-ovulatory aging and X-rays affect the male and female pronuclear chromatin structure after fertilization. These chromatin alterations could interact with DNA lesions induced in the spermatozoa prior to fertilization, such that development to first cleavage can be blocked.     

Fertilizing competency of multiple ovulated eggs in the domestic fowl (Gallus domesticus)

Fertilizing competency of multiple ovulated eggs in the domestic fowl was examined by fertilization in vitro and early development in culture. Normal laying hens (White Leghorn) were treated with 75 IU of PMSG for 7 days followed by injection of anterior pituitary extracts from chickens (CAPE). Ovulation began to occur 7.5 h after injection of CAPE. These hens ovulated 1-7 ova but some premature ovulation of GV stage ova were observed. In vitro fertilization of the multiple ovulated ova was examined by inseminating 10(6)-10(7) sperm onto the germinal disks in m-Ringer's solution. The gamete or zygote nuclei were detected by DNA specific fluorescence using DAPI (4',6'-diamidino-2-phenylindole) in the histological section prepared from the germinal disk. Process of fertilization was examined in the eggs incubated for 4 h after insemination in DMEM + liquid albumen at 41 degrees C under the atmosphere of 5% CO2 in air. Fertilization rate of the total multiple ovulated eggs was 55% (11/20), in which 90% (9/10) and 10% (1/10) in the eggs recovered 7.5-8.5 h and 9.0-9.5 h after CAPE injection were obtained, respectively. Normal pronuclei were formed in five eggs of those recovered 7.5-8.5 h after CAPE injection. Early development after fertilization in vitro was also examined by incubation for 12 h in DMEM + liquid albumen at 41 degrees C under the atmosphere of 5% CO2 in air. Although development in vitro was delayed compared to that in utero condition, normal development was observed in naturally and multiple ovulated eggs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the periovulatory period in superovulated heifers

Heifers (n=31) were superovulated with an FSH-P/cloprostenol regimen, and at 12 and 24 hours after the onset of estrus they were inseminated. Blood sampling for LH analyses and ultrasound scanning of the ovaries were performed at 4-hours intervals. The scanning, at which the first and last ovulations were recorded, was performed at 22.7 +/- 1.5 (mean +/- SD) and 31.0 +/- 1.5 hours after the LH peak, respectively. An average of 7.8 +/- 1.0 ovulations was monitored when the first ovulations were detected, while 2.8 +/- 0.7 ovulations occurred later. At 16 hours after detection of the first ovulations the oviducts were flushed and 5.6 +/- 0.5 fertilized and 2.3 +/- 0.3 unfertilized ova were isolated per animal. The fertilized ova displayed spherical pronuclei of synchronous development, and polyspermic penetration was not seen. At 24 hours after detection of the first ovulations the content of the remaining 3.3 +/- 0.5 nonovulatory follicles > 8 mm per animal was aspirated. Expanded cumulus investment was found in 69.4% of the oocytes, while 22.4% had abstricted the first polar body.     

Fertility of cooled and frozen rabbit sperm measured by competitive fertilization

The fertility of rabbit sperm that had been cooled to 5 degrees C or frozen and thawed was determined by competitive fertilization. Treatments were identified by labeling sperm with fluorescein isothiocyanate (FITC) or tetramethylrhodamine B isothiocyanate (TRITC). Sperm from different treatments were mixed and used in a competitive insemination experiment. Does were inseminated 5, 10 or 15 h prior to ovulation. Time of ovulation was controlled by injections of luteinizing hormone. The functional sperm transport, as determined by the number of sperm transported to the site of fertilization and capable of fertilizing oocytes, was estimated by counting the total number of differently stained sperm that surrounded or fertilized each oocyte. The fertility of sperm cooled to 5 degrees C was not affected (p less than 0.05) as compared to fertility of uncooled sperm. Functional sperm transport at all times of insemination and fertilization ratio at insemination 10 or 15 h before ovulation were reduced (p less than 0.05) for frozen-thawed vs. cooled sperm. No difference in fertilization ratio (p greater than 0.05) occurred, however, when does were inseminated 5 h before ovulation. While sperm survival and capacitation time appeared to play roles in fertility of frozen-thawed sperm, the most important factor was reduced functional sperm transport. However, fertility of frozen-thawed sperm was improved when the time from insemination to ovulation was reduced.     

Histone H1 kinase activity during in vitro fertilization of pig follicular oocytes matured in vitro

The present study was conducted to clarify the relationship between histone H1 kinase (H1K) activity and events associated with in vitro fertilization of pig follicular oocytes matured in vitro. Histone H1 kinase has been shown to be homologous with a maturation promoting factor (MPF). Cumulus-oocyte complexes obtained from prepubertal gilts were cultured for 46 h in a modified Waymouth's MB752/1 medium and were then inseminated in vitro with frozen-thawed and preincubated epididymal boar spermatozoa. At 4, 6, 8 and 10 h post insemination, the oocytes were stained with 10 microg/ml Hoechst-33342 and examined under a fluorescent microscope for the stage of fertilization, according to morphological changes of oocyte nuclear chromatin and the extent of sperm penetration. Sperm penetration was observed to occur within 4 h post insemination (20.5%), and the percentage of fertilized oocytes increased (P < 0.01) to 72.9% at 8 h post insemination. Pronuclear formation was observed from 6 h post insemination (3.3%) and the percentage increased (P < 0.01) to 46.8% at 10 h post insemination. In each examination period, H1K activities in unfertilized oocytes at metaphase-II remained unchanged (112.0 fmol/h/oocyte) and were higher (P < 0.01) than those in fertilized oocytes (30.1 fmol/h/oocyte). The H1K activity in fertilized oocytes such as oocytes emitting a second polar body, oocytes with an enlarging sperm head(s) and oocytes with multiple pronuclei did not differ significantly. These results suggest that MPF in pig oocytes is inactivated shortly after sperm penetration and is maintained at the basal level throughout pronuclear formation.     

A detailed analysis of early events during in-vitro fertilization of bovine follicular oocytes

Bovine follicular oocytes collected at slaughter were matured and fertilized in vitro with in-vitro capacitated spermatozoa. Analysis of 621 penetrated ova fixed at various times after in-vitro insemination led to definition of 6 stages of early development. A time sequence for sperm penetration, sperm head decondensation, male pronucleus formation, the activation of second meiotic division, female chromosome decondensation and pronucleus development was established. First sperm penetration into the ooplasm was recorded 6 h after insemination; 1-2 h was required for the sperm head to decondense and another 4-6 h to develop into the opposing pronucleus stage. Synkaryosis and first cleavage occurred 28 h after fertilization. Examination of the early stages revealed four types of abnormalities, i.e. polyspermy, polygyny, asynchrony between male and female pronucleus development, and preactivation of cytokinesis.     

Ovulation, fertilization and pronucleus development in superovulated gilts

Estrus was synchronized in 45 gilts by ingestion of Zinc-Methallibur in the feed for 15 d. On Day 16 each gilts was treated with PMSG (1200 IU i.m.) followed in 72 h by hCG (500 IU i.m.). Gilts were inseminated 24 and 36 h after the onset of estrus followed by slaughter of groups (n = 4 or 5) at 40 h, 44 h, 48 h, 52 h, 56 h, 60 h and 64 h after hCG injection. Ovaries were evaluated macroscopically and oocytes/embryos were recovered by flushing the oviducts. The ovulation rate increased from 38% to 87% from 40 to 45 h and remained constant thereafter. At 40 h, 36% of oocytes were penetrated by a single spermatozoon. The rate of fertilization increased from 36% (40 h) to 59% (44 h), to 65% (48 h), to 73% (52 h), to 76% (56 h), 80% (60 h) and to 64% (64 h). At 40 h all fertilized ova contained a decondensed sperm head. After another 4 to 8 h early pronuclei were common, and 52 h after hCG treatment opposed pronuclei were predominant. The first cleavages were recorded 64 h after hCG injection.     

Microfilaments during sea urchin fertilization: Fluorescence detection with rhodaminyl phalloidin

Rhodaminyl-labeled phalloidin is used to demonstrate the distribution of microfilaments during fertilization and early development in eggs of the sea urchins Arbacia punctulata and Lytechinus variegatus. The surface of unfertilized eggs have numerous punctate fluorescence sites at which rhodaminyl phalloidin binds, indicating the presence of actin oligomers or polymers. During fertilization this punctate pattern of fluorescence begins to change. Within thirty seconds of insemination, the fertilization cone is first detectable with this technique as an erect structure on the surface of the egg. The fertilization cone grows to a maximum size by 8–9 minutes, and is resorbed by 16 minutes after insemination. The surface of the fertilized egg displays numerous fluorescent fibers by 10 minutes after insemination rather than the punctate fluorescence observed in unfertilized eggs, indicative of the burst of microfilament assembly resulting in microvillar elongation. The elongated microfilaments persist through cytokinesis. Staining is also detected throughout the cortices of unfertilized, fertilized, and cleaving eggs. Cytochalasin E (10 μM, 30 min) prevents microfilament elongation and cytokinesis and reduces the cortical staining intensity after fertilization. At cleavage, contractile rings, appearing as narrow equatorial bundles of fibers, have been detected in Lytechinus variegatus as transient structures.     

Ovum recovery and in vitro fertilization in crab-eating monkeys (Macaca fascicularis)

A total of 301 oocytes were recovered from crab-eating monkeys and subjected to insemination in vitro resulting in two fertilized ova. Sixteen monkeys in 24 cycles received 37.5 IU of hMG daily from the second day of the menstrual cycle for 7 to 10 days. Oocytes were recovered under laparotomy at 20 to 49 hr after administration of 1,000–1,500 IU of hCG. The maturation rate of the recovered oocytes was 24.2% as judged from morphological criteria under the light microscope. With additional maturation culture, the rate increased to 36.2%. The matured oocytes were inseminated at 3 to 4 hr after aspiration using homologous spermatozoa which had been capacitated in vitro. Two oocytes were judged as being fertilized based on the presence of 3 and 5 pronuclei, respectively, when examined 12 hr after the insemination. This is the first report of in vitro fertilized ova in nonhuman primates in Japan.     

Transport of fertilised and unfertilized ova in sows

Transport of fertilised and unfertilized ova was studied in 22 crossbred (Landrace x Yorkshire) multiparous sows. Sows in the inseminated group (I-group, n=11) were inseminated once with 100ml of BTS extended semen from two fertile boars with a total of 10 x 10 (9) spermatozoa during the second oestrus after weaning between 18 and 8h prior to estimated time of ovulation, as estimated from the first oestrus after weaning. All the sows were slaughtered between 36 and 48 h after ovulation in the second oestrus after weaning by stunning and bleeding. After slaughter, the reproductive tract was immediately recovered, the isthmus was divided into three equal segments, and the number of ova was determined in each segment and in the upper third of the uterine horn from the UTJ. There were no significant differences (P>0.05) either in the intervals from ovulation to slaughter (42.3+/-6.2h versus 43.2+/-5.4h) or in the numbers of corpora lutea (CL) (18.2+/-5.5 versus 15.9+/-3.5) between the non-inseminated (N-group) and the inseminated groups (I-group), respectively. Ova recovery rate was 92.5% in the N-group and 82.9% in the I-group (P>0.05). In the I-group, ova had passed 2.2+/-0.3 segments whereas in the N-group, ova had passed 2.6+/-0.3 segments (P=0.38). It can be concluded that there is no difference in the transportation of either fertilised or unfertilized ova in the reproductive tract of pigs.     

Pronuclear synthesis of DNA in fertilized and parthenogenetically activated mouse eggs : A cytophotometric study

Mouse one-cell embryos were taken 1, 1.5, 2, 3, 4, 6, 8, 10, 13 and 18 h after insemination. One-cell parthenogenones were induced by treatment of mouse eggs obtained 20 h after HCG injection with hyaluronidase and cultured for 0.5, 1, 3, 4.5, 6, 8, 10, 12 and 24 h. Some parthenogenones were pulse-labelled with tritiated thymidine, cut and autoradiographed. Both the embryos and parthenogenones were Feulgen-stained, and integrated relative optical absorption of either pronuclei or nuclei of polar bodies was measured with a cytophotometer. In some fertilized eggs and parthenogenones the DNA synthesis sets in 4–6 h after either insemination or parthenogenetic stimulus. Between the 8th and 13th hour after insemination the fraction of DNA synthesizing embryonic pronuclei remained at the level 30–40%. Most parthenogenones duplicated their DNA content between the 8th and 12th hour after hyaluronidase treatment. The DNA synthesis time in pronuclei of embryos was determined to be 3.5–4.0 h and that of pronuclei of parthenogenones approx. 4 h. The minimal time of the G2 phase was estimated to be 3–5 h. The first labelled pronuclei of parthenogenones were detected 6 h after stimulus. Male pronuclei started and ended DNA synthesis earlier than female pronuclei. Differences in the DNA content between pronuclei of the parthenogenones (when there are two in one parthenogenone) were observed beginning with the 10th h after hyaluronidase treatment.The DNA content in the nuclei of the second polar bodies (PB) of embryos increased slowly between the 8th and 22nd hour after insemination, up to an overall value of 1.4 C. That of the nuclei of the polar bodies of parthenogenones accompanied the synthesis of DNA in pronuclei to the 10th hour after hyaluronidase treatment, up to an overall value of 1.4 C.     

Effect of time of ovulation and sperm concentration on fertilization rate in gilts

In normal production practices, sows and gilts are inseminated at least twice during estrus because the timing of ovulation is variable relative to the onset of estrus. The objective of this study was to determine if a normal fertilization rate could be achieved with a single insemination of low sperm number given at a precise interval relative to ovulation. Gilts (n=59) were randomly assigned to one of three treatment groups: low dose (LD; one insemination, 0.5 x 10(9) spermatozoa), high dose (HD; one insemination, 3 x 10(9) spermatozoa) or multiple dose (MD; two inseminations, 3 x 10(9) spermatozoa per insemination). Twice daily estrus detection (06:00 and 18:00 h) was performed using fenceline boar contact and backpressure testing. Transrectal ultrasonography was performed every 6 h beginning at the detection of the onset of standing estrus and continuing until ovulation. Gilts in the LD and HD groups were inseminated 22 h after detection of estrus; MD gilts received inseminations at 10 and 22 h after detection of estrus. Inseminations were administered by using an insemination catheter and semen was deposited into the cervix. The uterus was flushed on Day 5 after the onset of estrus and the number of corpora lutea, oocytes, and embryos were counted. Time of insemination relative to ovulation was designated as 40 to >24 h, 24 to >12 h, and 12 to 0 h before ovulation and >0 h after ovulation. The LD gilts had fewer embryos (P<0.04), more unfertilized oocytes (P<0.05) and a lower fertilization rate (P<0.07) compared to MD gilts. The effects of time of insemination relative to ovulation and the treatment by time interaction were not significant. We conclude that a cervical insemination with low spermatozoa concentration may not result in acceptable fertility even when precisely timed relative to ovulation.     

Immunocytochemical evidence for centrosomal phosphoproteins in mitotic sea urchin eggs

The change in distribution of centrosomal phosphoproteins was examined in sea urchin eggs from fertilization to the first cleavage by immunofluorescence staining with the anti-phosphoprotein antibodies, MPM-1 and MPM-2. The antibodies reacted with female pronuclei in unfertilized eggs as well as centriolar complexes located at the base of sperm flagella. After insemination, male and female pronuclei fused together to form a zygotic nucleus which was visualized by staining of fertilized eggs with the antiphosphoprotein antibodies. No major change in staining pattern was detected in extracted whole eggs until mitosis. As the fertilized eggs approached mitosis, however, the antigens started to redistribute from nuclei to the perinuclear position where the mitotic centrosomes were located. Detailed immunofluorescence observation of isolated spindles revealed that the phosphoantigens were retained in isolated structures. A major 225 kd polypeptide was recognized by the antibodies, suggesting that the 225 kd protein is a phosphocomponent of centrosomes. The area recognized by the antibody in mitotic poles enlarged with the progress of mitosis, suggesting that the antigens were apparently localized in the centrosphere. Centrospheres prepared from isolated spindles by salt extraction strongly reacted with the antibodies. One or two bright dots, which may represent centrioles, were visible in the isolated centrosphere. At the end of mitosis, the antigens again appeared in the newly formed daughter nuclei. Centriole-containing cytasters and centriole-free monasters were parthenogenetically induced in unfertilized eggs (Kuriyama and Borisy, (1983) J. Cell Sci. 61: 175-189). The antibodies stained centers of both the asters whether they contained centrioles or not, indicating that the antibodies recognizes the components of the pericentriolar material.     

From oocyte to embryo: a model, deduced from in vitro fertilization, for natural selection against chromosome abnormalities

A cytogenetical analysis was performed on 151 unfertilized oocytes, 22 fertilized eggs at the pronuclear stage, and 108 cleaved embryos obtained in the course of in vitro fertilization (IVF). Thirty-two per cent of unfertilized oocytes were abnormal, carrying nullisomies or disomies, mainly of D and G chromosomes, and a structural anomaly (Gq-) in one case. Fertilized eggs showed frequent asynchronism in the development of pronuclei and only 2 out of 8 karyotyped pronuclei were normal. Cleaved embryos were classified according to the number of pronuclei observed 17 hours after insemination. One per cent displayed a single pronucleus, and haploid chromosome complements were found in the corresponding cleaved embryos which were considered to be parthenotes. The rate of chromosome abnormalities of diploid eggs depended on their morphological aspect. Healthy cleaved embryos carried 12.5% of anomalies while this rate reached 37% in fragmented embryos (p less than 0.05). Lastly, 6% of fertilized eggs displayed three pronuclei or more. Only 41% of the corresponding embryos were triploid. Diploidy or diploidtriploid mosaicism were often encountered. This leads to a 21% rate of abnormalities in the preimplantation embryos. Parental karyotyping and HLA typing were carried out in a series of eight couples with in vitro idiopathic infertility or recurrent embryo degeneration in vitro. No abnormality was noted. According to these results, a model of natural selection of normal conceptuses is proposed.     

Xenogenous fertilization of laboratory and domestic animals in the oviduct of the pseudopregnant rabbit

Xenogenous fertilization was accomplished using bovine, porcine, and hamster follicular oocytes. The xenogenous fertilization rates for bovine and porcine follicular oocytes in the oviduct of the pseudopregnant rabbit were 13.4% and 2.0%, respectively. Temperatures of ovary, during transport to the laboratory, of 0 degrees or 37 degrees C had no effect on xenogenous fertilization rates of bovine oocytes. In vitro culture in 50 mug/ml FSH did not alter the xenogenous fertilization rates of bovine oocytes. Fertilization was observed with oocytes recovered 40 to 75 hr after insemination. Two cell embryos were recovered 70 to 75 hr after insemination. Ligation of the rabbit oviduct, number of ova deposited and sperm concentration did not affect the xenogenous fertilization rates of hamster ova. Cleavage of xenogenously fertilized hamster oocytes occurred between 28 and 29 hours after insemination.     

Inhibitory Effects of Diltiazem and Verapamil, Calcium Antagonists, on Fertilization-Induced Increase in the Phosphorylase Reaction in Echiuroid Eggs

The calcium antagonists diltiazem and verapamil at 100 μM caused considerable inhibition of the glycolysis system in recently fertilized eggs of the echiuroid, Urechis unicinctus . The levels of glycolytic intermediates in eggs were found to be higher 5 min after insemination than before fertilization while the levels of adenine nucleotides and inorganic phosphate were almost the same before and after fertilization. Addition of diltiazem or verapamil 30 sec after insemination did not inhibit fertilization, but resulted in maintenance of as low levels of glycolytic intermediates as in unfertilized eggs. The apparent mass action ratio in the phosphorylase step, calculated from the levles of glucose-1-phosphate and inorganic phosphate was normally higher in fertilized eggs than in unfertilized eggs, but was maintained at as low a level as in unfertilized eggs by adding these compounds 30 sec after insemination. Phosphorylase a activity also normally increased after insemination, but was maintained at a low level in fertilized eggs by adding these compounds. These compounds also inhibited the increased ⁴⁵Ca²⁺ uptake normally observed after fertilization. These results suggest that after fertilization, the Ca²⁺ level increases associated with fertilization-induced Ca²⁺ influx and that this stimulates Ca²⁺ dependent protein kinase to phosphorylate phosphorylase b , resulting in an increased rate of the phosphorylase reaction.     

Evidence for ovulation and fertilization in beef cows with short estrous cycles

Calves were weaned from 15 Polled hereford anestrous cows 25 to 42 days after calving. In eight cows the uterus was flushed on day 6 or 8 after the first postweaning estrus (day 0), and in seven cows the oviduct ipsilateral to the ovary containing an ovulation papilla was removed and flushed on day 3. One ovum (morula) was recovered from the eight uterine flushings, while six ova were recovered from six of the seven oviductal flushings. Of the six, three were fertilized (4 to 8 cells), two unfertilized and only the broken zona pellucida of one was recovered. An ovulation papilla was observed in all cows at the time of oviduct removal. Six of the 15 cows had cycles less than 12 days, and from four of those six fertilized ova were recovered. The data indicate that previously anestrous cows ovulate at their first postweaning estrus and the ova released are capable of being fertilized. Failure to maintain pregnancy appears to be due to early corpus luteum regression.     

The effect of time of intrauterine insemination on the development and viability of embryos collected from superovulated ewes

Eighteen Border Leicester x Scottish Blackface ewes, primed with 300 mg progesterone (12 d) and superovulated with decreasing doses (6, 5, 3 and 2 mg) of porcine FSH, were inseminated with fresh semen, using laparoscopic intrauterine procedures at 48 (Group E) or 60 h (Group L) after exogenous progesterone removal. Five days after insemination, embryos were collected and classified on the basis of their morphological development. During the subsequent 3 d of in vitro culture (38.5 degrees C; 5% CO2) the embryos were evaluated at 24-h intervals. After 72 h, the embryos were individually fixed (24 h) and stained with aceto-orcein and the nuclei were then counted to provide an objective index of cell proliferation and development. Mean (+/-SEM) ovulation rates for the 2 groups (9.2+/-1.5 and 7.1+/-1.2, respectively) and the corresponding percentages (53 vs 59) of embryos collected by laparoscopy were unaffected by insemination time. All donors yielded fertilized ova, but whereas all Group-E donors yielded 1 or more viable embryos (i.e., >32 cells), only 5 Group-L ewes yielded viable embryos (P<0.10). At collection, the percentages of embryos at the morula stage of development were 98 (Group E n = 44) and 39 (Group L n = 38; P<0.001). Few of the remaining ova (Group E = 0% Group L = 8%) were at the 1-cell stage of development when collected, indicating that retarded development post fertilization, not fertilization failure, was the principal consequence of delayed insemination. The percentages of embryos that continued to develop during in vitro culture were 91 and 37 for Groups E and L, respectively (P<0.001), and all of these reached the blastocyst stage. Of these blastocysts, 75 and 50% in Groups E and L hatched in vitro (P<0.10), with mean (+/-SEM) nuclei counts of 148+/-22.7 and 76+/-13.8 (P<0.02), respectively. In conclusion, while delayed intrauterine insemination did not affect the efficiency of ovum collection, it caused a major reduction in the yield of embryos that were capable of developing during in vitro culture. However, fertilization failure accounted for only 13% of the loss in viability following late insemination.