Development of centrifuged cow zygotes cultured in rabbit oviducts

Zygotes from superovulated cows were centrifuged and pronuclei were detected by differential interference-contrast microscopy in 73% of 106 zygotes. Zygotes were then transferred to ligated oviducts of follicular-phase, 1-day pseudopregnant or 7-day pseudopregnant rabbits and recovered 5 days later. Their development did not differ from that of uncentrifuged zygotes transferred to the opposite oviduct: 41% of the embryos recovered from rabbit oviducts contained 17-32 nuclei and an additional 5% contained greater than 32 nuclei. In another experiment, 399 ova from unmated cows were transferred to rabbit oviducts to determine whether centrifugation induced parthenogenetic development. After 7 days, 257 ova were recovered; 16% of the recovered ova had developed parthenogenetically and contained 2-30 nuclei. Neither centrifugation of the ova nor reproductive status of the rabbits influenced the proportion of parthenogenotes found. Parthenogenetic development was also observed in 14 of 71 ova (20%) recovered on Day 7 from uninseminated superovulated cows. In an attempt to increase the probability of detecting treatment differences, centrifuged and control cow zygotes were incubated for 7 (rather than 5) days in opposite oviducts of fourteen 1-day pseudopregnant rabbits. Development was unaffected by centrifugation: 61% of the zygotes recovered had developed beyond the 16-cell stage, with 23, 24 and 15% containing 17-32, 33-64, and greater than 64 nuclei, respectively. Taking into account the percentage of zygotes in which pronuclei can be seen, the recovery rate from rabbit oviducts, and the proportion of embryos that develop to the morula stage or beyond, 26% of the original group of zygotes would be candidates for transfer into recipient cows.     

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.     

Exposure of bovine embryos to trypsin during washing does not decrease embryonic survival

The objective of this study was to assess whether the exposure of zona pellucida-intact bovine embryos to the proteolytic enzyme, trypsin, during embryo washing has a detrimental effect on their subsequent survival and development. Embryos were collected nonsurgically from superovulated cows (n = 19) 7.5 d after insemination. Grade 1 and Grade 2 embryos were washed 12 times in modified Dulbecco's phosphate buffered saline (PBS) containing 0.4% bovine serum albumin (BSA), or in a series of five washes in BSA-PBS (without Ca++ and Mg++), two in 0.25% trypsin in Hank's solution (without Ca++ and Mg++) and five in PBS-BSA medium. Within 30 min after washing, embryos were either transferred nonsurgically into recipient cows, 7 to 8 d post estrus, or cryopreserved and transferred later. Frozen-thawed embryos from five of the donors were cultured for 72 h in vitro and their development was evaluated. Pregnancy rates did not differ (P>0.1) between recipient cows receiving control-washed and trypsin-washed embryos transferred fresh (51.0 vs 56.3%). However, pregnancy rates were higher (P<0.05) for frozen-thawed embryos treated with trypsin before cryopreservation than for frozen-thawed, control-washed embryos (68.2 vs 38.5%). Survival and development of embryos in vitro after cryopreservation did not differ between embryos subjected to the control- and trypsin-wash procedures. These results suggest that exposure of bovine embryos to trypsin for 2 to 3 min during washing did not have a detrimental effect on embryonic development, but may have enhanced cryopreservation of the embryos.     

Commercial aspects of bovine embryo transfer

Superovulated beef cows and heifers were nonsurgically collected 6 to 8 days post estrus. Commercial production results for 1976 through 1978 were 4979 pregnancies from 7814 embryos transferred for an overall pregnancy rate of 63%. In 1978, 519 superovulation procedures averaged 9.95 +/- 8.4 (S.D.) ova collected, 8.2 +/- 7.55 ova fertilized, 5.96 +/- 5.37 embryos transferred and 3.63 +/- 5.37 pregnancies per procedure. Embryos were transferred to recipient cows in estrus 12 hr before the donor (-12) the same time (0) or 12 hr after the donor (+12). The +12 group had a significantly lower pregnancy rate (61%, P<.05) than the 0 group (67%) or -12 group (66%). Transfer of early morula stage embryos resulted in a lower pregnancy rate (61%, P<.05) than late morula (67%) early blastocyst (67%) or late blastocyst (71%) stage embryos. A higher pregnancy rate (P<.05) was obtained with embryos of good morphological quality (71%) than with embryos graded fair and poor (55%). The pregnancy rate for embryos transferred nonsurgically was lower (44%) than the pregnancy rate for embryos transferred surgically during the same time period (66%). Pregnancy rates for three operators performing the nonsurgical transfers were 48%, 53%, 28%. No difference in pregnancy rate was found between embryos cultured 24 hr in BMOC-3 at 37C (62%) and embryos transferred the same day as collection (60%). Pregnancy rates for cultured embryos transferred to recipient cows in estrus 12, 24 or 36 hr after the donor were 68%, 62% and 60%, respectively. Embryos recovered on days 6, 7 and 8 were frozen in 1.5M DMSO and stored in liquid nitrogen several days to several weeks. Of 68 embryos frozen, 34 were viable post thaw. Upon transfer to recipient cows, the 34 viable embryos produced 23 confirmed pregnancies.     

Collection of tubal stage bovine embryos by means of endoscopy. A technique report

Here we describe the development and optimization of endoscopy-mediated transvaginal access for collecting ova and embryos from the bovine oviduct. The novel technique was developed in three experimental setups: In Experiment 1 embryos were collected unilaterally from nonstimulated heifers. We flushed the oviducts of superovulated heifers unilaterally (Experiment 2) and bilaterally (Experiment 3). In Experiment 1 the oviducts of 18 heifers were successfully cannulated, which resulted in the collection of twelve 1-cell to 8-cell embryos and one empty zona. Unilateral flushing of 13 animals (Experiment 2) resulted in 84 ova with 6.3 +/- 3.2 observed ovulation sites. Bilateral flushing of 25 animals (Experiment 3) resulted in 293 ova plus 10 empty zonae from 11.8 +/- 5.4 ovulation sites. Given our experience from these studies we optimized the technical equipment by improving the flushing metal catheter (Experiment 4). The novel catheter hermetically sealed the lumen of the ampulla at the moment, the medium was flushed through the oviduct. This resulted in a visible flow of medium via oviducts toward the embryo filter connected to an embryo flushing catheter that was fixed in the uterine horns. Our endoscopy-guided method is minimally invasive and facilitates the flushing of tubal stage embryos.     

The ovine uterus as a host for in vitro-produced bovine embryos

A series of experiments were conducted to determine whether bovine blastocysts would develop beyond the blastocyst stage in the ovine uterine environment. In Experiment 1, in vitro matured, fertilized and cultured (IVM/IVF/IVC) expanded bovine blastocysts were transferred into uteri of ewes on Day 7 or 9 of the estrous cycle and collected on Day 14 or 15 to determine if the bovine blastocysts would elongate and form an embryonic disk. Springtime trials with ewes that were synchronized with a medroxyprogesterone acetate (MAP) sponge resulted in a 78% blastocyst recovery rate, and 68% of the recovered spherical or elongated embryos had embryonic disks. In Experiment 2, transfer of 4-cell bovine embryos to the oviducts of ewes at Day 3 resulted in a lower recovery (47 vs 80%) than the transfer of blastocysts at Day 7 when embryos were recovered at Day 14. However, the percentage of embryos containing embryonic disks was higher for embryos transferred at the 4-cell stage (71%) than for embryos transferred as blastocysts (50%). In Experiment 3, IVF embryos from super-ovulated cows or Day 8 in vitro produced embryos transferred to cows were collected at Day 14 and were found to be similar in size to those produced by transfer to ewes in Experiment 2. In Experiment 4, the transfer of bovine blastocysts to ewes did not prolong the ovine estrous cycle. In Experiment 5, extension of the ovine estrous cycle by administration of a MAP releasing intravaginal device allowed bovine embryos to elongate extensively and to become filamentous. In Experiment 6, uterine flushings on Day 14 or Day 16 contained elevated levels of interferon-tau when bovine blastocyst were transferred on Day 7. Transfer of bovine embryos to the reproductive tract of a ewe allows some embryos to develop normally to advanced perimplantation stages and may be a useful tool for studying critical stages of embryo development and the developmental capacity of experimental embryos.     

Production of bovine identical twins via transfer of demi-embryos without zonae pellucidae

Embryos were collected nonsurgically from n?turally-cycling or superovulated donors 7 d after estrus. Forty-four morulae, early blastocysts and blastocysts classified as good to excellent were bisected using a fine glass needle to produce forty-four identical demi-embryos. The bisected demi-embryos, without zonae pellucidae, were nonsurgically transferred, either by twin or single transfer. An additional forty-eight embryos were collected from the same donors and transferred as a control. Among the twin transfers, 8 of the 13 recipients became pregnant (61.5%). Seven of them conceived twin fetuses (87%) and one a single fetus. However, only two sets of normal identical twin calves were obtained. Among the single transfers, 72.6% (45/62) of bisected embryos without zonae pellucidae resulted in pregnancy, of which 48.4% (30/62) were identical twins, and 24.2% (15/62) were singletons. Another 27.4% (17/62) of the recipients did not became pregnant. The pregnancy rate for whole embryos with zonae pellucidae was 72.9% (35/48). These data show that there was no significant difference between the pregnancy rates of bisected embryos without zonae pellucidae and whole embryos with zonae pellucidae transferred 7 d after estrus. Bisection of bovine embryos was simplified and even morula stage embryos were transferred without zonae pellucidae.     

Deep freezing of sheep embryos.

Sheep embryos, collected 1-8 days after oestrus, were placed in Dulbecco's phosphate-buffered saline medium (PBS). After treatment, the viability of the embryos was tested by temporary transfer to ligated rabbit oviducts. In Exp. 1, Days 5-8 embryos survived for at least 15 min at 0 degrees C in the presence of 1-5 M-DMSO. In Exp. 2, 12/14 Days 5-8 embryos survived after being frozen in 1-5 M-DMSO at 0-3 degrees C/min to temperatures ranging between-15 degrees and -60 degrees C and then thawed at 12 degrees C/min. In Exp. 3, Days 5-8 embryos were frozen in 1-5 M-DMSO at 0-3 degrees C/min to below-65 degrees C before being transferred to liquid nitrogen (-196 degrees C), and stored for 12 hr to 1 month. The embryos were thawed at 3 degrees C/min, 12 degrees C/MIN or 360 degrees C/min and, after transfer to rabbit oviducts, 0/4, 10/36 and 1/4, respectively, developed normally. The 11 embryos which were considered normal when recovered from the rabbit oviducts plus 1 slightly retarded embryo were transferred to 7 recipient ewes. Four ewes subsequently lambed, producing 5 lambs. In addition, 8 embryos were transferred to 4 ewes directly after thawing. Three of these ewes subsequently lambed, producing 3 lambs.     

Development and survival after transfer of cow embryos cultured from 1-2-cells to morulae or blastocysts in rabbit oviducts or in a simple medium with bovine oviduct epithelial cells

This study compares development of bovine 1-2-cell embryos in bovine oviduct epithelial cell co-culture (Group EC) with a glucose- and serum-free simple medium (CZB), or after surgical transfer to ligated oviducts of rabbits (Group RO). Embryos were surgically collected from superovulated donor cows 40-48 h after the beginning of oestrus and randomly distributed between the two groups. Embryos were cultured or incubated for 5 days. In Exp. 1, embryo quality scores and total numbers of cells in the two groups were compared. In Exp. 2, pairs of similarly treated morulae were transferred to each of 10 or 12 recipients in the Groups RO and EC, respectively. Total cell counts per embryo in both groups averaged 52 (P greater than 0.05), and the in-vitro culture system was equivalent to the rabbit oviducts in promoting embryo development for all characteristics measured. Embryo survival, as determined by ultrasound between Days 39 and 43 after oestrus, in 13 ideal recipients was 57% for embryos in Group EC and 58% for embryos Group RO. None of the 9 less desirable recipients was pregnant for either group. These results establish that cattle zygotes can develop to morulae in culture with bovine oviduct epithelial cells in a simple medium and can produce normal pregnancy rates.     

Superovulation of rabbits with FSH alters in vivo development of vitrified morulae

Morulae were flushed from the oviducts and uteri of New Zealand White (NZW) rabbits superovulated with either 6 (3 d) or 8 (4 d) injections of FSH and from non-superovulated controls. The percentages of embryos recovered from 4 d (100%, n = 8) donors was significantly higher (P < 0.01) than that of 3 d (76%, n = 16) and control (87%, n = 22) donors. Overall, fertilization rates were significantly lower for the 3 d embryos (P < 0.01). Most (86 to 90%) morulae were morphologically suitable for vitrification in an ethylene glycol-based solution. Following storage in liquid nitrogen, morulae were rapidly thawed and transferred to the uteri of pseudopregnant recipients. The total number of kits born for the 3 d, 4 d, and control groups was 40, 61 and 48, respectively. The percentage of live kits from morulae transferred was significantly lower for the 3 d (20%, n = 201) than either the 4 d (36%, n = 169; P < 0.01) or the control (31%, n = 157; P < 0.05) group. The mean number of kits born/recipient for the 3 d (2.4 +/- 2.9), 4 d (4.7 +/- 3.5), and control (3.0 +/- 2.2) protocols did not differ (P > 0.05). The estimated overall efficiency of producing kits based on normal morulae collected for control and 4 d groups, however, was nearly two-fold that for females given 6 FSH treatments. We conclude that the 4 d FSH superovulation regimen enhances the efficiency of rabbit reproductive biotechnology after embryo cryopreservation. These findings have important implications for rabbit colony management using embryo cryopreservation.     

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.     

Pregnancies after in-vitro fertilization of cow follicular oocytes, their incubation in rabbit oviduct and their transfer to the cow uterus

Cow embryos fertilized in vitro (1-8-cell) (n = 113) were transferred surgically to the ligated oviduct of pseudopregnant female rabbits (31 +/- 4 h after 75 i.u. hCG). Rabbits were killed 99 +/- 5 h later and 67 embryos were recovered: 45 (67%) had cleaved at least once, 15 had reached the morula stage and 2 blastocysts were obtained. Transfer of cleaved embryos (2-8-cell) led to a high recovery rate (84%) compared to 39% for one-cell embryos. Of the embryos incubated for more than 99 h in the rabbit oviduct, 41% were at the morula stage. Embryos incubated in vivo (n = 21) (8-cell to blastocyst) were transferred to the uterus of 14 synchronized recipient heifers by a surgical (N = 5) or a non-surgical (N = 9) procedure: 6 pregnancies resulted (4 from the non-surgical procedures). In addition, 27 (2-8-cell) cow embryos developed in vitro were transferred to the oviduct of synchronized heifers by a paralumbar surgical approach on a standing animal, but no pregnancies resulted. It is therefore concluded that (1) the rabbit oviduct can be used to obtain cow eggs at an embryonic stage sufficiently advanced to permit transfer to the uterus of a synchronized recipient; and (2) the pregnancy rate after in-vitro fertilization and incubation in the rabbit oviduct are similar to results with fertilization in vivo.     

Method for producing transgenic bovine embryos from in vitro matured and fertilized oocytes

Microinjection and in vitro culture procedures were developed to produce transgenic bovine embryos after in vitro fertilization of in vitro matured oocytes. In Experiment I, zygotes were subjected to pronuclear microinjection of DNA 18 or 24 h following addition of spermatozoa to oocytes. Microinjections were performed in either Hepes-buffered TCM-199 or modified Dulbecco's phosphate-buffered saline without glucose. Viability of embryos was similar at both injection times and for both media, as determined by morphological evaluation after culturing embryos in vitro for 10 d. In Experiment II, microinjected embryos were cultured 1) in rabbit oviducts, 2) in vitro in a 5% CO(2) in air, or 3) in a 5% CO(2) / 5% O(2) / 90% N(2) incubator. There were no significant differences between the 2 in vitro culture environments. The in vitro culture systems supported development of embryos significantly better than the rabbit oviducts; 33% of cleaved ova developed to blastocysts in vitro vs 10% in vivo; 98% of transferred ova were recovered from the rabbit oviducts. From both experiments, 6 of 92 blastocysts were positive for the microinjected DNA as determined by a polymerase chain reaction followed by gel electrophoresis.     

Superovulatory response of river bufalo (Bubalus bubalis )

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

First birth of an animal from an extinct subspecies (Capra pyrenaica pyrenaica) by cloning

Two experiments have been performed to clone the bucardo, an extinct wild goat. The karyoplasts were thawed fibroblasts derived from skin biopsies, obtained and cryopreserved in 1999 from the last living specimen, a female, which died in 2000. Cytoplasts were mature oocytes collected from the oviducts of superovulated domestic goats. Oocytes were enucleated and coupled to bucardo's fibroblasts by electrofusion. Reconstructed embryos were cultured for 36 h or 7 d and transferred to either Spanish ibex or hybrid (Spanish ibex male ×  domestic goat) synchronized recipients. Embryos were placed, according to their developmental stage, into the oviduct or into the uterine horn ipsilateral to an ovulated ovary. Pregnancy was monitored through their plasmatic PAG levels. In Experiment 1, 285 embryos were reconstructed and 30 of them were transferred at the 3- to 6-cells stage to 5 recipients. The remaining embryos were further cultured to day 7, and 24 of them transferred at compact morula/blastocyst stage to 8 recipients. In Experiment 2, 154 reconstructed embryos were transferred to 44 recipients at the 3- to 6-cells stage. Pregnancies were attained in 0/8 and 7/49 of the uterine and oviduct-transferred recipients, respectively. One recipient maintained pregnancy to term, displaying very high PAG levels. One morphologically normal bucardo female was obtained by caesarean section. The newborn died some minutes after birth due to physical defects in lungs. Nuclear DNA confirmed that the clone was genetically identical to the bucardo's donor cells. To our knowledge, this is the first animal born from an extinct subspecies.     

In vitro development of DNA-injected embryos co-cultured with goat oviduct epithelial cells in Korean native goats (Capra hircus aegagrus)

In vitro development of Korean native goat embryos was investigated in 2 different culture systems with and without goat oviduct epithelial cells (GOEC). Estrus was synchronized by inserting intravaginal progestagen-impnegnated sponge (Veramix) containing 60 mg medroxyprogesterone acetate (MAP) for 14 d. Superovulation was induced with follicle stimulating hormone (FSH). Goat ova were surgically obtained by retrograde flushing the oviducts of does at 66 to 68 h after MAP removal. Mean number of recovered ova per doe was 7.28 +/- 3.91, and the proportion of fertilized embryos in recovered ova was 66.5% (121/182 ). Fertilized embryos were cultured for 9 d in CR1aa medium supplemented with 10% estrous goat serum (EGS) at 38.5 degrees C, 5% CO(2) in air. There was no difference in development of the embryos to the morula stage between the 2 culture systems (84.4 and 84.0%, respectively). However, developmental rate to blastocysts (65.6%) of the embryos co-cultured with GOEC was significantly higher than of those (12.0%) cultured without GOEC (P < 0.001). Goat zygotes were injected with bovine beta-casein/human lactoferrin cDNA fusion gene (pBL1). When the DNA-injected embryos were co-cultured with GOEC, developmental rates of the embryos to the morula and blastocyst stages were 82.9 and 36.6%, respectively. The results obtained in this study indicate that "blocking" of in vitro development of Korean native goat embryos appears to occur at the morula stage, but can be overcome to some extent by co-culture with GOEC. In the co-culture system, DNA-injected goat embryos could successfully develop to normal hatching blastocysts.     

Use of PMSG antiserum in superovulated cattle?

Two Pregnant Mare Serum Gonadotrophin (PMSG) antisera were tested in 174 dairy cows that were superovulated with PMSG and were then given prostaglandin at 60 hours after PMSG. At 48 hours after injection of prostaglandin, the cows were given either PMSG antiserum (monoclonal (n=56) or polyclonal (n=57)), or saline as control (n=61). Ova (n=1,206) were recovered either nonsurgically or after slaughter. Of these, 757 were evaluated morphologically to be transferable embryos. A proportion of these embryos (n=295 from 52 flushed donors) were transferred to synchronized recipients and the pregnancy results were recorded. The reproductive function of 37 flushed donors was followed for 6 months after superovulation. No significant effect of the PMSG antisera could be demonstrated in any of the parameters studied (i.e., ovulation rate, number of follicles at collection, total yield of ova, fertilization rate, number of transferable embryos, pregnancy results after transfer of embryos, or period required by the donor cows for restitution of reproductive function after superovulation and recovery). It is concluded that use of PMSG antiserum did not improve the embryo yield in terms of the number and quality of transferable embryos or enhance normalization of reproductive function of the donor in the 6-month period after superovulation. Therefore, in an embryo transfer operation, the routine use of PMSG antiserum in a PMSG superovulation regimen in cattle is not recommended.     

Early pregnancy associated thrombocytopenia as an initial response to pregnancy in cattle

This study was conducted to determine if early pregnancy-associated thrombocytopenia exists in cattle as has been demonstrated in mice and in humans. Three experiments were designed to compare peripheral platelet counts in pregnant versus nonpregnant animals. In Experiment 1 heifers (n = 25) were artificially inseminated 12 h after the onset of estrus. Peripheral platelet counts in 19 pregnant versus 6 nonpregnant heifers did not reveal any significant differences between groups after insemination. In Experiment 2 embryos were collected nonsurgically from superovulated cows (n =18) on Days 6 to 7 after estrus. Platelet counts were monitored every 12 h after the first insemination until 60 h after the second insemination. Platelet counts and the number of embryos collected nonsurgically from these superovulated donors did not show any significant correlations (P>0.05). Ten recipient heifers synchronized to donor animals received either an unfertilized ovum or a good quality embryo via nonsurgical transfer into the uterus. There were no significant reductions in platelet counts after transfer. In Experiment 3 platelet counts were monitored daily in four pregnant and five nonpregnant recipient heifers between Day 0 and Day 30 after embryo transfer on Day 8 of the cycle. The platelet counts did not reveal any significant differences between the pregnant and nonpregnant groups throughout Days 0 to 30. These results indicate that early pregnancy-associated thrombocytopenia cannot be demonstrated in cattle. Peripheral platelet counts cannot be used as an indicator of early pregnancy in cattle.     

Development of porcine ova that were centrifuged to permit visualization of pronuclei and nuclei

The obscured pronuclei or nuclei in living one- and two-celled pig ova were revealed after centrifugation for 3 min at 15,000 X g. To determine viability of centrifuged ova, one- and two-celled pig ova were collected from superovulated gilts; half of the ova were centrifuged and all ova were transferred into recipient gilts. Prior to transfer all embryos were stained with tetramethylrhodamine isothiocyanate (TRITC) to distinguish the experimental embryos from the recipients's own ova. Centrifuged ova were transferred into one oviduct of recipient gilts and uncentrifuged ova were deposited into the opposite oviduct. Embryos were recovered 4 days after transfer and were stained with lacmoid or Hoechst 33342 to assess their stage of development. Centrifugation had no detectable influence on survival of the recovered embryos to 4 days. Centrifugation is a simple, reliable method for revealing pronuclei and nuclei of one- and two-celled pig ova and apparently does not alter subsequent preimplantation development.     

Bovine somatotropin increases embryonic development in superovulated cows and improves post-transfer pregnancy rates when given to lactating recipient cows

Previous studies indicated that the use of bovine somatotropin (bST) in concurrence with a timed artificial insemination (TAI) protocol increased pregnancy rates. However, the mechanisms for such a bST effect on fertility were not clear. Objectives of this study were to determine the effects of bST on fertilization and early embryonic development after cows received a superovulation treatment, test whether embryos recovered from bST-treated cows were more likely to survive after transfer to recipients, and evaluate whether treatment of recipient cows with bST affects pregnancy rates. Lactating (n = 8) and nonlactating (n = 4) Holstein donor cows were superovulated, inseminated at detected estrus and assigned to a nontreated control group or to a treatment group receiving a single injection of bST (500 mg, sc) at insemination. Embryos were nonsurgically flushed 7 days after AI and frozen in ethylene glycol for direct transfer. Embryos derived from bST-treated (bST-embryos) or control (control-embryos) donors were transferred to lactating Holstein recipient cows that received either bST treatment 1 day after estrus (500 mg, sc; bST-recipients) or were untreated controls (control-recipients). Thus, there were four treatment groups: control-embryos/control-recipients (n = 43), bST-embryos/control-recipients (n = 41), control-embryos/bST-recipients (n = 37), and bST-embryos/bST-recipients (n = 60). Pregnancy was determined by palpation per rectum 33-43 days after embryo transfer. Unfertilized ova per flush was less for bST than for control (1.0 +/- 0.9 < 3.7 +/- 0.9; P < 0.04). Percentage of transferable embryos was greater for bST than for control (77.2% > 56.4%; P < 0.01). Number of blastocysts per flush was greater for bST than for control (2.4 +/- 0.7 > 0.4 +/- 0.7; P < 0.04). Pregnancy rates following embryo transfer were 25.6% for control-recipient/control-embryo, 43.2% for bST-recipient/control-embryo, 56.1% for control-recipient/bST-embryo, and 43.3% for bST-recipient/bST-embryo. Transfer of bST-embryos increased pregnancy rates compared with transfer of control-embryos (P < 0.04). An interaction between embryo and recipient treatments (P < 0.05) indicated that treatment of recipient cows with bST increased pregnancy rates as compared to control-recipients that received a control-embryo. However, there was no additive effect when bST-recipients received a bST-embryo. Administration of bST at AI decreased the number of unfertilized ova, increased the percentage of transferable embryos, and stimulated embryonic development to the blastocyst stage. Moreover, bST affected both early embryonic development and recipient components to increase pregnancy rates following embryo transfer.