In vitro fertilization of ova from cows experimentally infected with a non-cytopathic strain of bovine viral diarrhea virus

Bovine viral diarrhea virus infection was induced in 16 heifers by inoculation of a noncytopathic strain of bovine viral diarrhea virus (BVDV). Six BVDV-free heifers served as controls. On Day 8 after inoculation, cumulus—oocyte complexes were collected from ovaries of animals at the second peak of fever preceded by leukopenia. The oocytes were then matured and fertilized in vitro. There was no significant difference (48% vs. 54% P>0.05) in the percentage of cleaved oocytes between infected and non-infected animals. However, the proportion of embryos that developed to the blastocyst stage was significantly higher for the control group than for BVDV group (29% vs. 14%) (P<0.01). All follicular fluids and cumulus—oocyte complexes collected from infected animals tested positive for the presence of the virus, but embryos produced by in vitro fertilization 7 days after in vitro co-culture tested negative.     

The role of Haemophilus somnus in early embryonic death. I. The effect of the organism on embryos by day 8 postbreeding

A study using 23 healthy, mature, virgin Holstein-Friesian heifers was designed to determine if H. somnus caused detrimental effects in early bovine embryos and the mechanism(s) that induced these effects. Superovulated heifers were artificially inseminated 12 and 24 h after standing estrus using highquality, Haemophilus-free semen from a single ejaculate of one bull. Treatment heifers (n=12) were exposed by intrauterine infusion 12 h after the second insemination to approximately 1.5 x 10(9)H. somnus organisms (Iowa strain 1229) suspended in 10 ml of sterile 0.85% phosphate buffered saline (PBS). Control heifers (n=11) were inseminated and the infused with sterile PBS. Embryos were recovered 8 d after the second insemination using non-surgical technique and evaluated microscopically and graded on their estimated survivability. Representative embryos were also examined for in vitro culture survival time, histopathological changes, vital stain uptake and bacterial contamination. Following embryo recovery, uterine flush solution was centrifuged at 10,000 x G. Sediment was submitted for bacteriologic examination and supernatant preserved for quantitation of H. somnus immunoglobulins. Results to date indicate that H. somnus had a detrimental effect on early bovine embryos. H. somnus was recovered from the tissues of one treated animal. Significantly more (P 相似文献   

Normal reproductive capacity of heifers that originated from in vitro fertilized embryos cultured with an antiviral compound

Bovine viral diarrhea virus (BVDV) can associate with in vitro fertilized (IVF) bovine embryos despite washing and trypsin treatment. An antiviral compound, DB606 (2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan), inhibits the replication of BVDV in bovine uterine tubal epithelial cells, Madin Darby bovine kidney cells, and fetal fibroblast cells. As well, DB606 in in vitro culture medium does not affect embryonic development. Antiviral-treated-IVF embryos placed into recipients developed into clinically normal calves. The objective of this project was to determine if these resultant heifer calves were capable of reproducing. Seven heifers from each of the treatment groups (natural breeding, IVF embryo, and IVF embryo cultured in DB606) of the previous study were used. At 20-27 months of age, the heifers were exposed to a fertile bull in a single pasture during a 63 d breeding season. Five of the seven heifers originating from natural breeding were pregnant 35 d after removal of the bull and calved. All of the heifers resulting from transfer of untreated IVF embryos were pregnant at 35 d; however, one aborted the fetus at 5-7 months of gestation. All of the heifers derived from transfer of IVF embryos cultured in DB606 were pregnant and calved. Offspring from dams of all treatment groups were clinically normal at birth. Adjusted 205 d weaning weights were not significantly different among the offspring of the treated and untreated dams. These results indicate that culture of bovine-IVF embryos in DB606 does not impair future reproductive capacity of resulting heifers.     

The effect of bovine pestivirus infection on the superovulatory response of Friesian heifers

The pathogenesis of reproductive loss associated with bovine pestivirus infection during the preovulatory period was investigated using superovulated heifers. Twenty-five Friesian heifers were selected and randomly assigned to either a control group (n = 12) which did not become infected or to a treatment group (n = 13) which became infected following intranasal instillation of 2 ml of serum inoculum containing 5.5 log(10) TCID(50)/ml non-cytopathic virus, 9 d prior to artificial insemination (AI). Transrectal ultrasonography was used to monitor follicular development and ovulation during the superovulatory period. Animals were superovulated using a standard protocol of twice-daily injections of FSH-P and then were inseminated twice commencing 12 h after the onset of estrus. The intensity of expression of estrus was higher in the control heifers than in the pestivirus-infected heifers. Of 13 pestivirus-infected heifers, only 3 heifers displayed standing estrus compared with that in the control group, in which 10 of 12 heifers exhibited standing estrus. The mean number of ova/embryos recovered from the control group heifers was 5.75 +/-2.31, of which 4.00 +/- 0.72 were evaluated as transferable quality embryos. In comparison, heifers in the pestivirus-infected group yielded only a mean of 0.60 +/-0.34 ova/embryos, of which 0.23 +/- 0.22 were transferable quality embryos. Based on ultrasonographic examination, 24 h after the first AI 82% of the presumptive ovulatory follicles had ovulated in the control group compared with an ovulation rate of only 17% in the treated group. The results of this experiment demonstrated that bovine pestivirus infection during the preovulatory period could adversely affect ovulation, thus leading to a significant reduction in the number of palpable corpora lutea and in the number and quality of embryos recovered.     

Collection of two-cell pig embryos and their culture in media containing different protein components

Eggs were flushed from the oviducts of slaughtered gilts that had been inseminated after synchronization of estrus and ovulation with pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG). Of 347 eggs collected at a recovery rate of 73.8%, 41.8% had not cleaved by 32 h after expected ovulation time. Of those cleaved, 86.9% were at the two-cell stage.Two-cell embryos were cultured in Dulbecco's medium containing either no protein or 20% of one of the following: lyophilized bovine serum albumin, lyophilized fetal calf serum or heat-inactivated serum from slaughter heifers, rabbits or barrows. Dulbecco's medium without protein did not support further development of embryos. Addition of heat-inactivated blood serum from slaughter heifers, rabbits or barrows resulted in development rates similar to those obtained by using commercially available products. Optimal embryonic development rates of 54.5% were obtained with addition of heat-inactivated bovine serum. Of the two-cell embryos only three (2.1%) developed past the four-cell stage in these culture media.     

Effect of human leukemia inhibitory factor on in vitro development of parthenogenetic bovine morulae

The present study was conducted to investigate the effect of human leukemia inhibitory factor (hLIF) addition to synthetic oviduct fluid medium (SOFM) supplemented with human serum (HS) on the development of in vitro matured and parthenogenetically activated bovine oocytes. The oocytes matured for 30 h were exposured to ethanol (7%, 7 min) and cytochalasin B (5 mug/ml, 5 to 6 h). The treated oocytes were cultured for 5 d in SOFM supplemented with HS, and Day-5 morulae were cultured for 2 d in SOFM supplemented with HS and with or without hLIF (5000 U/ml) to investigate the subsequent in vitro development to the blastocyst stage. Of the 1531 oocytes that were parthenogenetically activated, 592 (37.5%) cleaved to the 2- to 8-cell stage and 174 (13.8%) developed to the morula stage. The addition of hLIF at the morula stage resulted in a significantly (P<0.01) higher rate of development to the blastocyst stage in the medium with hLIF (55.9%) than without hLIF (28.9%). The mean cell number per blastocyst developed in the medium with hLIF was also significantly (P<0.01) higher than that developed in the medium without hLIF. To evaluate the viability, 6 parthenogenetically developed blastocysts were transferred to 3 recipient heifers (2 embryos per heifer), while in 2 other recipient heifers estrus was prolonged after transfer. The plasma progesterone levels of the 2 recipient heifers at the 28th day after transfer were 8.1 ng/ml and 9.0 ng/ml, but pregnancy was not observed by ultrasonic scanning. The present results indicate that the addition of hLIF to in vitro-produced, Day-5 parthenogenetic bovine morulae significantly improves the subsequent development to the blastocysts stage; however, the present method still does not promote for development of parthenogenetic fetuses in cattle.     

Nuclear transplantation in the bovine embryo: assessment of donor nuclei and recipient oocyte

Blastomeres from 2- to 32-cell bovine embryos were transferred to enucleated oocytes matured either in vivo or in vitro by micromanipulation and electrofusion. The percentage of donor cells fusing with the recipient oocytes was dependent on relative cell size or stage of development. Therefore, when smaller donor karyoplasts (17- to 32-cell vs. 2- to 8-cell) were transferred, the rate of fusion was significantly less (p less than 0.01). After fusion, nuclear transfer embryos were cultured either in vitro or in vivo (in a ligated ovine oviduct). Nuclear transfer embryos cultured in vitro developed to the 4- to 6-cell stage after 72 h (4-cell, 71%; 8-cell, 33%, 16-cell, 33%; p less than 0.30), whereas nuclear transfer embryos cultured in vivo developed to the morula or blastocyst stage (2- to 8-cell, 11.7%; 9- to 16-cell, 16.0%; 17- to 32-cell, 8.3%; p greater than 0.30) after 4 or 5 days. Freshly ovulated oocytes (collected 36 h after the onset of estrus), when used as recipients, resulted in morula/blastocyst-stage embryos more often than in vitro-matured oocytes or in vivo-matured oocytes collected 48 h after the onset of estrus (20% vs. 7.8% and 6.7%, respectively; p less than 0.02). After in vivo culture, nuclear transfer embryos were mounted and fixed or transferred nonsurgically to the uteri of 6- to 8-day postestrus heifers. Seven pregnancies resulted from the transfer of 19 embryos into 13 heifers; 2 heifers completed pregnancy with the birth of live calves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early cycle FSH-p priming as a prelude to superovulating gonadotropin administration in ewes and heifers

The effect of an exogenous FSH treatment in the periovulatory, post-LH surge period on superovulatory response in the subsequent cycle of ewes and heifers was investigated. Thirty-five ewes were synchronized with progestagen pessaries and pregnant mares serum gonadotropin. The day following the onset of estrus (Day 1) 17 ewes received one intramuscular injection of 5 mg follicle stimulating hormone of porcine origin (FSH-p). All 35 ewes received another progestagen pessary on Day 1 and were superovulated with horse anterior pituitary extract (HAP). The ewes were bred and embryos collected 6 days following the onset of estrus. Early cycle FSH-p administration did not increase the subsequent ovulation rate (6.5 vs. 8.4 for controls, n.s.). Recovery rate for the FSH-p treated animals was higher (78.5% vs. 49.3%; P<0.05) as was fertilization rate (100% vs. 62.4%; P<0.05). The final result was a mean of 4.4 transferable embryos per ewe treated among the FSH-p boosted ewes and 2.6 transferable embryos per ewe treated among the control ewes.Twenty-nine heifers were brought into estrus with one 500-μg injection of prostaglandin F_2α (PG). Twelve of the 29 heifers were given one intramuscular injection of 10 mg FSH-p on either Day 2 or 3 (Day 1 is the day following the onset of estrus). All heifers were superovulated starting on Day 11–16, over a 4-day period using a decreasing dosage of FSH-p. Prostaglandin was administered at the time of the fifth superovulatory FSH-p injection and the heifers were bred by artificial insemination. Ova were recovered between 2 and 4.5 days following the onset of estrus. There was no effect on ovulation rate due to the interval from FSH-p priming to the day of superovulatory FSH-p initiation. The proportion of heifers that ovulated when given a FSH-p injection early in the cycle was higher than in the control group (94% vs. 68%; P<0.05). The primed heifers had a higher number of ovulations than did the control heifers (16.3 vs. 6.2; P<0.01). The effect of higher ovulation rate carried through all parameters measured, so that the FSH-p primed heifers also had a higher number of fertilized ova than the controls (10.7 vs. 3.9; P<0.05), indicating that there was no significant deterioration in ovum quality due to the FSH-p priming. The results show that FSH-p improved superovulatory efficiency in both sheep and cattle.     

Sanitary status of oocytes and embryos collected from heifers experimentally exposed to Leptospira borgpetersenii serovar hardjobovis

In a preliminary trial and three experiments, a total of 30 Holstein heifers were experimentally infected with a culture of Leptospira borgpetersenii serovar hardjobovis via one or more routes (uterine, cervical supraconjunctival, intranasal) and oviductal and uterine fluids recovered post-mortem or in vivo following superovulation with FSH. All routes of administration were effective in establishing Leptospira infection in the reproductive tract and Leptospira were identified in the oviductal and uterine fluids of all 30 heifers by microscopy. The incidence of infection was confirmed by positive identification of serum antibodies by the microscopic agglutination test (MAT). Twenty-one samples of the embryos (n=59) recovered were cultured using bacteriological procedures and all tested negative for the infectious microorganism. Using polymerase chain reaction (PCR) assay, however, showed that 29% (7/24) of morula and blastocyst stage embryos, and one out of 29 oocytes tested positive for the presence of leptospiral DNA. A single oocyte or embryo collected from the infected heifers was inoculated intravenously to 26 test heifers. None of the test heifers developed antibody titers to Leptospira. It was concluded that, despite the presence of leptospires in the reproductive tract of donor animals and the association of leptospiral DNA with uterine stage embryos, the transmission of this disease is unlikely to occur by transfer of in vivo produced embryos in the bovine.     

Influence of uterine flushings from superovulated cows on in vitro bovine morulae development

To evaluate early embryo development, 248 good to excellent bovine morulae were cultured in Ham's F-10 medium, supplemented with 10% steer serum, uterine flushings from Days 6, 10 or 15 following estrus (0.01, 0.1, 1.0 and 10% protein; 64 mg protein/ml), and 1.0% uterine flushings and 10% steer serum. Final development scores for embryos in steer serum were significantly higher (range across experiments was: 4.06 to 4.37) than for embryos cultured in uterine flushings alone (-0.23 to 0.52). Treatment means were not different (P >0.05) when 10% steer serum was added to 1.0% uterine flushings. A higher percentage of embryos in 10% steer serum (92%) than in 10% steer serum plus 1.0% uterine flushing from Day 6 (33%), Day 10 (45%) and Day 15 (50%) developed to hatched blastocysts. Embryos cultured in 1.0% Day 6 uterine flushings plus 10% steer serum required more time to attain the early blastocyst and blastocyst stages, while embryos in 1.0% Day 15 uterine flushings and 10% steer serum developed at the same rate as controls to the expanded blastocyst stage, but hatched sooner (72.8 vs 96.5 h). These results suggest substance(s) in uterine secretions can have inhibitory and stimulatory influences on early bovine embryo development.     

Culture of one- and two-cell bovine embryos to the blastocyst stage in the ovine oviduct

The ovine oviduct was evaluated as a culture system for early bovine embryos. One- to two-cell embryos were collected from superovulated heifers killed 36 or 48 h after the onset of estrus, embedded in agar cylinders, and transferred to oviducts ligated at the uterotubal junction. After 5 d (6.5 to 7.0 d after donor estrus), embryos were recovered and evaluated for development to the late morula or blastocyst stage. In Experiment 1, 86 embryos were cultured in 10 ewes in which the onset of estrus was synchronized with that of the donors. Fifty-eight embryos (68%) were recovered; of these, 31 (53%) had continued normal development. In Experiment 2, development in ovariectomized versus intact cyclic ewes was compared. Recovery from ovariectomized ewes (26/39, 67%) did not differ from intact cyclic ewes (26/35, 74%) and the proportion developing normally also did not differ (ovariectomized: 7/26, 27%; intact cyclic: 11/26, 42%). In Experiment 3, embryo development was compared in anestrous versus ovariectomized ewes. Recovery rate (anestrous: 22/43, 51%; ovariectomized: 20/51, 39%) and the proportion developing normally (anestrous: 8/22, 37%; ovariectomized: 9/20, 45%) did not differ between treatments. Developmental competence of oviduct-cultured embryos was tested by transfer to 16 synchronous heifers, of which eight (50%) became pregnant; five delivered calves. Results indicate that the ovine oviduct provides an adequate site for the culture of early bovine embryos.     

Meiosis in Bovine Oocytes Matured in Vitro and in Vivo

Meiosis in bovine oocytes has; been studied after maturation in vitro or in vivo. Oocytes for in vitro maturation were collected from the ovaries of slaughtered cattle without regard to the phase of the estrous cycle while in vivo maturation was studied in oocytes from gonadotrophin-stimulated heifers at times varying between 6 and 36 h after the beginning of behavioural estrus. Oocytes from slaughtered cattle were classified according to their cumulus complex and ooplasm and were cultured for 6, 12, 18, 24, 36 or 48 h in modified Krebs-Ringer bicarbonate buffer before fixation) for cytogenetic analysis. Oocytes from stimulated heifers were aspirated from follicles or flushed from the oviducts, classified according to cumulus and ooplasm, and fixed within 6 h of collection. Nuclear maturation was more rapid in vitro than in vivo. The largest proportion of oocytes reached maturity (Mil) after 12 to 18 h in culture or 30 to 36 h after the onset of behavioural estrus. Oocytes devoid of cumulus cells or showing signs of vacuolation or degeneration had virtually no capacity for nuclear maturation.     

Effects of progesterone or estradiol on uterine tubal transport of ova in the cow

An experiment was designed to determine the effect of progesterone (P) or estradiol benzoate (EB) on uterine tubal transport of ova in the cow. Intramuscular injections of P, EB, or corn oil (C) were administered to heifers 24 hours after the end of estrus. The heifers were euthanatized 60 hours after the end of estrus and the location of the ovum or zygote was determined. Venous serum levels of progesterone and estradiol-17beta were measured by radioimmunoassay. The mean uterine tube (UT) length was 23.9 cm. An ovum or zygote was recovered from 11 of 14 heifers. Serum levels of progesterone and estradiol-17beta were above normal bovine levels following the P and EB treatments, respectively. The mean UT ovum transport rates were 0.42, 0.21 and 0.23 cm/hour in the P, EB and C treatment groups, respectively. The UT ovum transport rate was increased (P<0.05) by the P treatment and EB treatment had no effect (P > 0.05) when compared with the C treatment.     

Embryonic development in superovulated dairy cattle exposed to elevated ambient temperatures between Days 1 to 7 post insemination

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 degrees 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 F(2)alpha (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 degrees C) or under hyperthermic conditions (daily exposure up to 16 h at 30 degrees C and 8 h at 42 degrees 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.     

Birth of double-muscled Belgian Blue calves after transfer of in vitro produced embryos into dairy cattle

The possible application of the bovine in vitro fertilization technique for economical beef production was evaluated by transferring in vitro produced Belgian Blue embryos to synchronized dairy cows and heifers. In total, 4167 oocytes, collected in the slaughterhouse from double-muscled Belgian Blue cows, were matured in vitro. Frozen-thawed semen from 3 Belgian Blue bulls was used for in vitro fertilization. Zygotes were cultured in B(2) + 10% estrous cow serum together with oviductal cells at 39 degrees C in 5% CO(2) in air. After 7 days, 576 (13.8%) transferable embryos were obtained. One hundred and eighteen of the most advanced embryos were selected for fresh transfer into 90 recipients. Some of the remaining embryos were frozen using conventional methods. After fresh transfer, 50 recipients (55.6%) had elevated progesterone at day 23. Thirty cows (33.3%) calved after a mean gestation length of 282.8+/-6.0 days and produced 25 single births and 5 twins. The sex ratio was 71.4%. The mean birth weight was 45.1+/-8.3 kg. Three calves were of the conventional type instead of double-muscled and 2 calves died of congenital malformations. After transfer of in vitro produced frozen-thawed Belgian Blue embryos into 27 recipients (1 embryo/recipient), 2 bull calves (7.4%) were born. Bovine embryo production by in vitro techniques could form a low-cost supply of beef calves. However, to render it commercially attractive, selection of sires and dams has to be performed with great care.     

Factors affecting in vivo viability of DNA-injected bovine blastocysts produced in vitro

In vitro matured and fertilized bovine ova were microinjected with pBL1, which consisted of the bovine beta-casein gene promoter, human lactoferrin cDNA and SV40 polyadenylation signal. Of the 2931 zygotes injected, 2505 (85.5%) survived 1 h after DNA injection and were cultured in 50-microl drops of CR1aa medium containing 3 mg/ml BSA under mineral oil at 39 degrees C, 5% CO2 in air. Cleaved (2- to 8-cell) embryos were selected at approximately 48 h after DNA injection and then cultured further in 50-microl drops of CR1aa medium supplemented with 10% (v/v) FBS. Blastocysts were classified into 4 quality grades and 3 developmental stages by morphological criteria. Then all but poor quality blastocysts were nonsurgically transferred to the uterus of heifers 7 to 8 d after natural estrus. Following transfer, the recipients were observed for signs of estrus, and pregnancy was confirmed by palpation per rectum at approximately 60 d of gestation. Although 72.0% (1804/2505 ) of the DNA-injected zygotes reached 2- to 8-cell stages only 5.2% (131/2505) developed to blastocysts. A total of 75 DNA-injected, in vitro cultured blastocysts were transferred to 59 recipients. When 2 blastocysts were transferred to a single recipient, only the better quality embryo was counted. The overall pregnancy rate was 30.5% (18/59 ) and reflected 1) an apparent correlation between the quality of embryos and the pregnancy rate. However, the difference was not statistically significant. 2) expanded blastocysts had a higher pregnancy rate (50.0%, 11/22 ) than early (13.3%, 2 15 ) or mid (22.7%, 5/22 ) blastocysts with a significant difference between expanded and early blastocysts (P < 0.05). 3) the pregnancy rate of DNA-injected blastocysts was higher when they were transferred at Day 7 (34.5%, 10/29 ) or 8 (36.8%, 7/19 ) than at Day 6 (9.0%, 1/11 ). The results indicate that the developmental stage of DNA-injected bovine embryos may be one of contributing factors in improving the pregnancy rate after transfer, although the effects of the quality and culture period of the embryos may not be inconsequential.     

Production efficiency of Japanese black calves by transfer of bovine embryos produced in vitro

The objective of this study was to examine the production efficiency of Japanese Black beef calves after transfer of bovine embryos derived from an in vitro procedure. In vitro-produced (IVP) embryos were obtained from in vitro maturation and fertilization and in vitro development by co-culture with cumulus cells until 7 or 8 days after insemination. In vivo-developed (IVD) embryos from superovulated Japanese Black heifers and cows 7 days after artificial insemination were used as a control group. Bovine embryos were transferred nonsurgically to recipient cows on Day 7 +/- 1 of the estrous cycle. Pregnancy was diagnosed by palpation per rectum at Day 60 to 70 after estrus. Pregnancy, abortion, perinatal accident and birth rates were examined according to the origin of embryos (IVP or IVD), the number of transferred embryos (single or twin) and the storage status (fresh or frozen-thawed). In Experiment 1, production efficiency by twin transfer of fresh IVP embryos was examined. Higher pregnancy rates (52 1% vs 42 9%, P < 0.05) and birth rates (47.0% vs. 33.0%, P < 0.05) were obtained by twin transfer than by single transfer of fresh IVP embryos. Thus, the twin transfer of fresh IVP embryos was effective for production of calves, although the birth rates for single and twin transfers of fresh IVD embryos were still higher (55.5% and 76.1%, P < 0.05). But the abortion and perinatal accident rates for twin transfer of fresh IVP embryos were also significantly greater than those for single and twin transfer of fresh IVD embryos (P < 0.05). In Experiment 2, production efficiency by twin transfer of frozen-thawed IVP embryos was examined. Either single or twin transfer of frozen-thawed IVP embryos resulted in a similar pregnancy rate (41.3% vs. 46.7%, P > 0.05) and birth rate (34.1% vs. 41.1%, P>0.05). Thus, in combination with frozen-thawed IVP embryos, the twin transfer did not enhance production efficiency. In conclusion, Japanese Black beef calves could effectively produce calves by twin transfer to Holstein recipients when using fresh IVP embryos, and by single transfer when using frozen-thawed IVP embryos.     

Agreement among evaluators of bovine embryos produced in vivo or in vitro

Six experienced individuals evaluated 40 embryos on videotape for stage of development and quality grade. These 40 observations comprised 15 embryos produced in vivo, 15 embryos produced in vitro, and 10 embryos that were repeated throughout the videotape. Embryos produced in vivo were recovered from uterine flushings of superovulated heifers 7 d after estrus, and embryos produced in vitro were harvested 7 d after insemination of in vitro-matured oocytes. Embryos of various stages (morulae, blastocysts, or degenerated) and quality grades (1 = excellent, 2 = good, 3 = fair, 4 = degenerated) were recorded on videotape for evaluation. After video microscopy, the embryos were stained and the number of nuclei per embryo was counted. Six evaluators reviewed the videotape and the percentage of agreement and kappa (k; agreement beyond chance) among evaluators were determined for classifications of stage and grade. Consistency of each evaluator's responses was estimated using the 10 repeated embryos. Agreement within evaluators was higher for stage of embryo development (89.2%) than quality grade (68.5%). Agreement among evaluators for stage was slightly higher with embryos produced in vivo (85.0%, k = 0.74) than in vitro (72.3%, k = 0.48). Agreement among evaluators for grade was similar with embryos from in vivo (61.0%, k = 0.46) and in vitro (57.7%, k = 0.42) production. For both sources of embryos, agreement was substantially better for Grades 1 and 4 than for Grades 2 and 3. The results of this study suggest that good to excellent agreement exists for classifying Day 7 bovine embryos by stage and by extremes of quality grade (Grades 1 and 4) but not by degree of abnormal morphology (Grades 2 and 3). Simple grading criteria of Grade 1 (highest quality), Grade 2 (morphologic defects), and Grade 3 (degenerated) maximized agreement among evaluators.     

Embryos derived from the in vitro fertilization of oocytes of pregnant cows

The aim of our study was to determine if the oocytes of pregnant cattle are capable for undergoing embryonic growth following in vitro fertilization. The ovaries of nine heifers at 4 to 7 months of pregnancy were collected at an abattoir and transferred to the laboratory. A total 191 oocytes (10.6 per ovary) collected by aspiration were matured and fertilized by frozen-thawed semen. Embryos were co-cultured with granulosa cells in modified TCM 199 medium and 20% estrous cow serum. The cleavage rate of embryos was 48%, and 41% of of the cleaved embryos developed to the morula/blastocyst stage 7 days after insemination. Additionally, the ovaries of 10 nonpregnant heifers were also collected, yielding 213 oocytes (10.7 per ovary). The cleavage rate was 51%, and 35% of those which cleaved reached the morula/blastocyst stage. No significant differences were found between the two groups. The average number of transferable-stage embryos obtained from pregnant and nonpregnant animals was 4.1 and 3.7, respectively. Our results indicate that preganancy does not influence the meiotic competence of bovine oocytes, and transferable stage embryos can be obtained by the fertilization of oocytes derived from pregnant animals.     

Effects of passive immunization against inhibin on ovulation rate and embryo recovery in holstein heifers

The effects of acute neutralization of endogenous inhibin on ovulation rate and circulating FSH levels were investigated. Nine or ten days after estrus, 5 heifers were given a single injection of 75 ml iv inhibin antiserum produced in a castrated male goat, while another 5 were given the same amount of a castrated male goat serum. All heifers were given injections of PGF2alpha im at 48 h and 60 h after the serum injection. Those exhibiting an estrus were artificially inseminated with frozen-thawed semen. Seven or eight days after the insemination, ova or embryos were collected using a non-surgical method. Administration of inhibin antiserum resulted in a significant increase in the number of medium-sized follicles compared with the number in the control animals. The number of large follicles in the inhibin-neutralized animals was 4.8 +/- 2.4 (mean +/- SEM; n = 5) on the day of estrus, while there was a single large follicles in the ovaries of control animals. Seven or eight days after estrus, 3 to 16 ova or embryos were recovered from 4 of 5 animals, and 64 % of the total ova/embryos were transferable. Administration of inhibin antiserum produced a significant increase in the concentrations of plasma FSH from 12 to 72 h after the serum injection compared with the levels in the control animals (P < 0.05). After the onset of estrus, preovulatory LH and FSH surges were noted in inhibin-neutralized animals and magnitude of the rise in each hormone was similar to the control animals. The present study demonstrates that a single injection of the inhibin antiserum induces multiple ovulations probably by enhancing FSH secretion, and that recovery of embryos is equal to that observation after an ordinary FSH treatment.