Unfertilized Ova Research Articles

Degenerative Disorders of the Central Nervous System

Degenerative Disorders of the Central Nervous System

Legendary Kinase Controls Early Mitoses

Chinese legend has it that Pan Gu broke out of an egg, created the heavens and earth out of chaos (and egg parts), and stood on the earth to hold up the heavens. At the end of the Western 20th century, Pan Gu has been reborn as a serine-threonine kinase. The DNA replication block that follows meiosis is derepressed at fertilization, but just how the unfertilized ovum is prevented from entering S phase is largely a mystery. Fenger et al. have identified and characterized Pan Gu (PNG), which controls the onset of embryonic DNA synthesis in Drosophila . png is expressed only in the maternal ovaries and in embryos up to 3 hours after fertilization. Because plutonium ( plu ) mutants can enhance the phenotype evinced by weak png mutations (aberrant DNA synthesis and mitoses), the authors asked whether PNG and PLU (an ankyrin-repeat protein) associate. PNG immunoprecipitated with PLU; however, association was dependent on PNG kinase activity, suggesting that phosphorylation or the wild-type conformation of PNG was essential for coprecipitation. Additionally, association of PNG and PLU appeared indirect and was most likely mediated by an unknown adaptor protein, as PNG and PLU failed to associate in yeast two-hybrid experiments. PNG stabilized the levels of cyclin A and B, and led to higher levels of histone phosphorylation, presumably mediated by the kinase CDC2. These results suggest a mechanism whereby S phase is suppressed: by keeping mitotic kinases active (which prevent the activation of DNA replication origins), and maintaining chromatin in a highly condensed state (through histone phosphorylation), PNG may control the switch from mitosis to DNA synthesis in early Drosophila embryos. Fenger, D.D., Carminati, J.L., Burney-Sigman, D.L., Kashevsky, H., Dines, J.L., Elfring, L.K., and Orr-Weaver, T.L. (2000). PAN GU: A protein kinase that inhibits S phase and promotes mitosis in early Drosophila development. Development 127 : 4763-4774. [Online Journal]

Developmental capacity of cumulus oocyte complexes collected from prepubertal cattle with and without gonadotropin stimulation employing ultrasound-guided follicular aspiration

A total of 139 laparoscopies and 106 uterine flushings were performed in 28 female sheep of three different breeds (Finnish Landrace, Merino, German Blackface). Nine 1-year-old Finnish Landrace and German Blackface sheep were used as recipients. Oestrus was synchronised by intravaginal sponges and the donors were superovulated using either 16 mg follicle stimulating hormone (FSH-P) or 1500 IU pregnant mares' serum gonadotrophin (PMSG). Embryo collections were done by laparoscopy through three stab incisions in the area anterior to the udder. A uterine horn was punctured close to the uterine body and a flexible catheter was inserted into the tip of the uterine horn. It was necessary to clamp the tip of the uterine horn with grasping forceps and flush the uterine lumen twice to obtain high recovery rates. Superovulatory treatment led to an average of 4.7 ± 4.6 corpora lutea (CL) without differences between PMSG and FSH-P. A total of 226 ovine embryos were recovered of which 144 (63.7%) were fertilised, including 65 (28.8%) damaged morulae. The remaining portion consisted of 26.5% unfertilised ova and 9.7% empty zonae pellucidae. The embryo recovery rate using laparoscopic technology was approximately 75%. A repeated embryo recovery was possible up to three times on a monthly basis with an efficiency of approximately 75–80%. The fourth flush resulted in a significantly reduced recovery rate (47.4%). The limiting factor for continued laparoscopic embryo collection was the postoperative protrusion of the endometrium at the puncture wounds of the uterine walls. Laparoscopic transfer of eight freshly collected ovine embryos to four recipients led to one pregnancy. Transfer of ten frozen/thawed embryos to five recipients led to three pregnancies. All pregnancies resulted in the delivery of normal lambs.

Structure of the avian oviduct with emphasis on sperm storage in poultry

The macroanatomy, histology, and fine structure of the avian oviduct is reviewed and related to its role in fertile egg production. The avian oviduct functions as a biological assembly line, beginning sequentially with the deposition of the albumen around the fertilized or unfertilized ovum, then the shell membrane, and lastly, the shell, all within 25 hr of ovulation. While in transit through the oviduct, the fertilized ovum progresses to the pre-gastrulation stage of development.

Superovulatory response of Colombian Creole cattle to two doses of FSH

On Day 3 of the estrous cycle (estrus = Day 0), dairy heifers were given either 10 mg i.m. FSH-P (FSH-P primed; n = 9) or a saline vehicle (saline primed; n = 9). On Day 10, all heifers were superovulated with FSH-P (total = 27.7 mg i.m.) in declining doses over 5 d. Heifers were inseminated artificially at estrus. From Day 2 until estrus, the number and size of follicles >2 mm were monitored daily by ultrasonography. The mean (± SEM) number of corpora lutea (CL) (6.2 ± 1.5 vs 10.7 ± 0.9; P<0.05) and the mean number of recovered embryos and unfertilized ova (3.6 ± 1.7 vs 8.4 ± 2.2; P<0.05) were lower in FSH-P-primed than in saline-primed heifers. Prior to initiation of superovulation, follicles >10 mm appeared on Days 6 to 7 in saline-primed heifers but only on Days 8 to 10 in FSH-P-primed heifers (P<0.05). Also, until Day 10, the mean number of follicles 4 to 6 mm and 7 to 10 mm was higher (P<0.05) in FSH-P-primed than in saline-primed heifers. After initiation of the superovulatory treatment (Day 10 to estrus), saline-primed heifers had a greater and faster increase in the mean number of follicles >10 mm (P<0.02) than FSH-P-primed heifers did. Depletion in the number of follicles 2 to 3 mm (P<0.001) between Day 10 and estrus and in the number of follicles 4 to 6 mm (P<0.05) between Day 12 and estrus occurred in both groups of heifers. Decreased superovulatory response and embryo recovery in FSH-P-primed heifers may have been due to the presence of large follicles (>10 mm) prior to the initiation of the superovulatory treatment which reduced the ability of small follicles to grow into larger size classes during superovulatory treatment.

Use of gonadotropin releasing hormone (GnRH) and prostaglandin F 2α (PGF 2α) to synchronize ovulations in superstimulated cattle

Holstein heifers (n = 29) were used to determine whether thermal stress during the first 7 d of embryonic development may increase the incidence of embryonic abnormalities in dairy cattle. Heifers were acclimated to environmental chambers at 20°C for 9 d and superovulated with follicle stimulating hormone-pituitary (FSH-P; 40 mg total), beginning on Days 9 to 11 of the estrous cycle. Prostaglandin F2α (Lutalyse; 50 mg total) was administered on Day 3 of FSH-P. Heifers were inseminated artificially at estrus and then maintained at either thermal neutrality (20°C) or under hyperthermic conditions (daily exposure up to 16 h at 30°C and 8 h at 42°C) for 7 d beginning at 30 h after the onset of estrus. On Day 7 post estrus, embryos were recovered nonsurgically and evaluated morphologically for stage of development and quality. The distribution of embryos classified as normal, abnormal, retarded or as unfertilized ova, differed (P<0.001) between heat stress and thermoneutral treatments. Only 20.7% of 82 embryos recovered from stressed heifers were normal compared with 51.5% of 68 embryos from thermoneutral animals. Stressed heifers had a higher incidence of abnormal and retarded embryos with degenerate nonviable blastomeres. Responses indicated that thermal stress from 30 h after the onset of estrus to Day 7 post estrus increases the incidence of abnormal and retarded embryos in superovulated heifers.

Protein Intake and Development of Ovarian Follicles and Embryos of Superovulated Nonlactating Dairy Cows

Nonlactating Holstein cows (n=12) were assigned randomly to one of two diets (approximately 30% concentrate) in a crossover design. Urea and soybean meal were used to increase CP content of diet from 12.3 to 27.4%. Mean concentrations of plasma urea were 9.8 and 21.3mg/dl for respective diets. After 35 d on diets, cows were brought to a synchronized estrus (progestin implant plus PGF2α, injection). From d 10.5 to 14.5 following estrus, cows received i.m. injections of decreasing doses of FSH to initiate superovulation. On d 3 of FSH treatment, PGF2α was injected with FSH. Cows were bred twice by AI when detected in estrus. Embryos were collected nonsurgically 6.5 d after breeding, and quality was assessed using visual, microscopic, and staining techniques. Development of follicles throughout this period was monitored using ultrasonography. Using ultrasonography, no differences were detected in the number or percentages of preovulatory, anovulatory, and ovulatory follicles induced during superovulation. Number of recovered embryos averaged eight per cow. Numbers and percentages of normal embryos, abnormal or retarded embryos, and unfertilized ova were similar between diets, as were the numbers and percentages of transferable and nontransferable embryos. Excess intake of CP failed to affect the growth or number of ovarian follicles and health and number of embryos of energy adequate, nonlactating dairy cows.

Ultrastructural studies of sperm penetration in the egg of the European catfish,Silurus glanis

The process of sperm penetration into the egg of the European catfish Silurus glanis L. was investigated by scanning and transmission electron microscopy. Unfertilized ovum membrane under the internal micropylar aperture shows a critical depression with undulation and short microvilli corresponding to a specialized sperm entry site structure. Twenty seconds after insemination, the fertilizing spermatozoon fused with the egg at this specialized structure, without formation of an earlier fertilization cone, which had been confirmed in eggs of other fish. A cytoplasmic eminence developed however, immediately under the fused sperm head and then disappeared with time after fertilization to form a circular area around the fused sperm head, which in turn became embedded in the circular area with shortening of the flagellum. The micropyle in the catfish egg consisted of a micropylar vestibule and a canal, the internal micropylar canal being wide enough to permit the penetration of only one spermatozoon.

The effect of dose schedule and route of administration on superovulatory response to folltropin in holstein cows

Two commercial FSH products were compared in a retrospective study on 3990 commercial superovulations and embryo recoveries in dairy heifers and cows. In addition, the 56-day nonreturn rate of 19,400 embryos produced with these two preparations was analyzed. Embryo collections were performed during a 16-year period from donors of Holstein and Ayrshire breeds. Folltropin (Vetoquinol S.A., Lure cedex, France) group (Group F) consisted of 2592 superovulations, of which 80% were performed on heifers and 20% on cows, and Pluset (Laboratorios Calier, S.A., Barcelona, Spain) group (Group P) of 1398 treatments, of which 66% and 34% were on heifers and cows, respectively. Total number of recovered structures, number of transferable embryos, and the proportion of unfertilized ova (UFO) and degenerated embryos were analyzed. Distribution of embryos into quality grades (1–3) and developmental stages (4–9) according to the IETS classification guidelines and means for each collection were evaluated. The proportion of low-responders having fewer than five corpora lutea and yielding fewer than five embryos or ova was investigated for each treatment. Group P yielded 1.1 recovered structures more than Group F (P < 0.001). Consequently, however, the number of transferable embryos did not differ among the groups, being 7.0 and 7.1 in Groups F and P, respectively. Instead, there was an increase in the number of UFO from 2.0 in Group F to 3.0 in Group P (P < 0.001). The quality of embryos and the developmental stages were similar between the groups and there was no difference in the proportion of low-responding donors in Group F and Group P. Also, there was no difference in the nonreturn rate after transfer of embryos originating from donors superovulated with Folltropin or Pluset. It was concluded that equal numbers of transferable embryos and pregnancies can be achieved with Folltropin and Pluset.

Influence of embryonic secretory chemicals on selective oviductal transport in mares

SummaryEquine embryos are selectively transported from oviducts to uteri from 5 days 10 h to 5 days 22 h after ovulation. Selective transport appears to be initiated by a characteristic of equine oviductal embryos that is not present in unfertilised ova. To test the hypothesis that selective oviductal transport is initiated by stage‐specific secretion of embryonic chemicals, experiments were completed to identify chemicals whose secretion by embryos was temporally associated with oviductal transport, and which initiated oviductal changes that were consistent with oviductal transport. Day‐6 embryos incubated in vitro secreted minimal amounts of proteins, steroids and prostaglandin F2α, and relatively high amounts of prostaglandin E2 (PGE2). Embryonic PGE2 secretion was not detected on Day 3 or Day 4, was first detected on Day 5 and in larger amounts on Day 6. PGE2 bound specifically to equine oviducts on Days 2 and 5. Uterine entry of oviductal embryos was hastened in 6 of 11 mares by intraoviductal PGE2 treatment, but was not hastened by saline vehicle treatment or no treatment. These results support the hypothesis that embryonic PGE2 initiates oviductal transport in mares.

Sex determination of porcine preimplantation embryos via y‐chromosome specific DNA sequences

Abstract Sex of preimplantation porcine embryos was determined by DNA amplification using porcine male(Y chromosome)‐specific DNA primers in the polymerase chain reaction (PCR). In order to determine the sensitivity of this sexing method, single porcine embryos ranging from unfertilized ova to the blastocyst stage were amplified in the PCR using the Y‐specific primers, and analyzed by ethidium bromide‐staining of polyacrylamide gels. The 192 bp product which denotes the presence of the Y chromosome was seen in the embryos. The unfertilized ova which is of female origin gave no product. These results are representative of PCR analysis of a total of 34 swine embryos. Results obtained using the PCR for sexing were validated by karyotyping and confirmed by in situ hybridization with the porcine Y‐chromosome‐specific probe. In order to confirm the sex of the embryos determined by PCR, 10 day‐old porcine preimplantation embryos were biopsied to produce a small number of cells for sex determination via PCR, whil...

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.

The effect of treatment with bovine somatotropin (BST) on the superovulatory response of cattle

The objective of this study was to evaluate the utilization of gonadotropin releasing hormone (GnRH) as part of a superovulatory regimen for Zebu cattle. Forty Zebu cows were superovulated with 40 mg of follicle stimulating hormone-pituitary (FSH-P) divided in eight fractions of 5 mg injected at 12-h intervals. Luteolysis was induced with 15 mg of luprostiol injected at 48 h after the first injection of FSH-P. Half of the animals were injected with 200 ug of GnRH 3 h after the onset of standing estrus. The other 20 animals were not injected with GnRH. All the cows were inseminated three times at 12-h intervals, starting at the time of standing estrus. Embryos were recovered nonsurgically 7 d after the last insemination.Palpation per rectum performed immediately after collection of the embryos did not show differences in the number of corpora lutea between groups (P > 0.05). Likewise, there were no significant differences between treatments with respect to the total number of embryos plus ova, total number of embryos, or the number of transferable embryos recovered (P>0.05). The number of blastocysts, morulae, degenerated morulae and unfertilized ova was similar for the two groups.It is concluded that the incorporation of GnRH into a part of the superovulatory treatment for Zebu cattle does not improve the results of such treatment.

Noninvasive measurement of glucose uptake by preimplantation sheep embryos and unfertilized ova

Using a noninvasive, fluorometric assay, glucose uptake was measured in individual preimplantation sheep embryos and unfertilized ova. Glucose uptake remained low through the morula stage of development. By the late morula stage, glucose uptake began to increase and was significantly higher by the blastocyst stage ( P < 0.08). This pattern was similar to that reported in mice.

Collection and transfer of ovine embryos by laparoscopy

A total of 139 laparoscopies and 106 uterine flushings were performed in 28 female sheep of three different breeds (Finnish Landrace, Merino, German Blackface). Nine 1-year-old Finnish Landrace and German Blackface sheep were used as recipients. Oestrus was synchronised by intravaginal sponges and the donors were superovulated using either 16 mg follicle stimulating hormone (FSH-P) or 1500 IU pregnant mares' serum gonadotrophin (PMSG). Embryo collections were done by laparoscopy through three stab incisions in the area anterior to the udder. A uterine horn was punctured close to the uterine body and a flexible catheter was inserted into the tip of the uterine horn. It was necessary to clamp the tip of the uterine horn with grasping forceps and flush the uterine lumen twice to obtain high recovery rates. Superovulatory treatment led to an average of 4.7 ± 4.6 corpora lutea (CL) without differences between PMSG and FSH-P. A total of 226 ovine embryos were recovered of which 144 (63.7%) were fertilised, including 65 (28.8%) damaged morulae. The remaining portion consisted of 26.5% unfertilised ova and 9.7% empty zonae pellucidae. The embryo recovery rate using laparoscopic technology was approximately 75%. A repeated embryo recovery was possible up to three times on a monthly basis with an efficiency of approximately 75–80%. The fourth flush resulted in a significantly reduced recovery rate (47.4%). The limiting factor for continued laparoscopic embryo collection was the postoperative protrusion of the endometrium at the puncture wounds of the uterine walls. Laparoscopic transfer of eight freshly collected ovine embryos to four recipients led to one pregnancy. Transfer of ten frozen/thawed embryos to five recipients led to three pregnancies. All pregnancies resulted in the delivery of normal lambs.

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 < 0.025) than in controls at metestrus. On the other hand, when diestrus rats were injected with PGE 1 (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 PGE 1 (p < 0.05) than PGE 2 or PGF 2α. The same pattern of PG production was observed with incubated embryos, but in this case the amount of PGE 1 released was significantly higher (p < 0.01) that the PGE 1 released by unfertilized ova. The importance of PGE 1 released by fertilized and unfertilized ova in the differential ovum transport in pregnant and cycling rats is discussed.

Noninvasive measurement of pyruvate uptake by ovine preimplantation embryos and unfertilized ova

A noninvasive, fluorometric assay was used to measure pyruvate uptake by ovine unfertilized ova and preimplantation embryos up to the expanded blastocyst stage. There was a significant (P<0.02) increase in pyruvate uptake between unfertilized ova and one-cell embryos, after which uptake levels declined. Another significant increase (P<0.02) was seen at the early morula stage, with uptake levels remaining high through the expanded blastocyst stage. The pattern of pyruvate uptake observed was markedly different from that which has been reported in mice. These data suggest that the metabolic events of preimplantation embryonic development may differ substantially between the mouse and sheep.

Superovulation fails to increase human blastocyst yield after uterine lavage

Uterine lavage affords the potential for non-invasive human blastocyst recovery, with obvious potential for preimplantation genetic diagnosis. In an effort to duplicate in women the multiple blastocyst recovery per cycle that can be achieved in several other species, we initiated a programme in which fertile women underwent superovulation, followed by lavage and embryo collection. We superovulated 15 fertile women, aged 21-40, in 29 cycles using one of four regimens. Insemination was by either intercourse or artificial intracervical donor insemination with cryopreserved sperm from men of proven fertility. In 28 of 29 cycles, the uterus was lavaged daily for 1, 2, or 3 days between 5 and 10 days after human chorionic gonadotropin (hCG) administration or luteinizing hormone (LH) surge. Almost total fluid volume was recovered in every lavage. There were no retained pregnancies and no complications. Surprisingly, only two morulae, one blastocyst, and four unfertilized ova were recovered. Thus, alterations in ovulation induction, insemination timing, or lavage techniques must be contemplated in order to increase the blastocyst yield and thus fulfil the potential of uterine lavage for preimplantation diagnosis.

Evaluation of the incorporation of GnRH into a superovulatory regimen for Zebu cattle

The objective of this study was to evaluate the utilization of gonadotropin releasing hormone (GnRH) as part of a superovulatory regimen for Zebu cattle. Forty Zebu cows were superovulated with 40 mg of follicle stimulating hormone-pituitary (FSH-P) divided in eight fractions of 5 mg injected at 12-h intervals. Luteolysis was induced with 15 mg of luprostiol injected at 48 h after the first injection of FSH-P. Half of the animals were injected with 200 ug of GnRH 3 h after the onset of standing estrus. The other 20 animals were not injected with GnRH. All the cows were inseminated three times at 12-h intervals, starting at the time of standing estrus. Embryos were recovered nonsurgically 7 d after the last insemination. Palpation per rectum performed immediately after collection of the embryos did not show differences in the number of corpora lutea between groups (P > 0.05). Likewise, there were no significant differences between treatments with respect to the total number of embryos plus ova, total number of embryos, or the number of transferable embryos recovered (P>0.05). The number of blastocysts, morulae, degenerated morulae and unfertilized ova was similar for the two groups. It is concluded that the incorporation of GnRH into a part of the superovulatory treatment for Zebu cattle does not improve the results of such treatment.

The effects of dominant follicular regression and FSH priming on the superovulatory response of cattle

On Day 3 of the estrous cycle (estrus = Day 0), dairy heifers were given either 10 mg i.m. FSH-P (FSH-P primed; n = 9) or a saline vehicle (saline primed; n = 9). On Day 10, all heifers were superovulated with FSH-P (total = 27.7 mg i.m.) in declining doses over 5 d. Heifers were inseminated artificially at estrus. From Day 2 until estrus, the number and size of follicles >2 mm were monitored daily by ultrasonography. The mean (± SEM) number of corpora lutea (CL) (6.2 ± 1.5 vs 10.7 ± 0.9; P<0.05) and the mean number of recovered embryos and unfertilized ova (3.6 ± 1.7 vs 8.4 ± 2.2; P<0.05) were lower in FSH-P-primed than in saline-primed heifers. Prior to initiation of superovulation, follicles >10 mm appeared on Days 6 to 7 in saline-primed heifers but only on Days 8 to 10 in FSH-P-primed heifers (P<0.05). Also, until Day 10, the mean number of follicles 4 to 6 mm and 7 to 10 mm was higher (P<0.05) in FSH-P-primed than in saline-primed heifers. After initiation of the superovulatory treatment (Day 10 to estrus), saline-primed heifers had a greater and faster increase in the mean number of follicles >10 mm (P<0.02) than FSH-P-primed heifers did. Depletion in the number of follicles 2 to 3 mm (P<0.001) between Day 10 and estrus and in the number of follicles 4 to 6 mm (P<0.05) between Day 12 and estrus occurred in both groups of heifers. Decreased superovulatory response and embryo recovery in FSH-P-primed heifers may have been due to the presence of large follicles (>10 mm) prior to the initiation of the superovulatory treatment which reduced the ability of small follicles to grow into larger size classes during superovulatory treatment.