Non-surgical Transfer Research Articles

Effect of sucrose concentration used for one-step dilution upon in vitro and in vivo survival of bovine embryos refrigerated in glycerol and 1, 2-propanediol

Bovine embryos collected on Day 7 of pregnancy were suspended in 1.4 M glycerol or 1.6 M 1, 2-propanediol solution. Embryos were loaded into 0.25-ml plastic straws in phosphate buffered saline (PBS) containing 0, 0.2, 0.4 or 0.8 M sucrose. The straws were placed directly into a cooling chamber and cooled from 0°C to −6°C at 1°C/min and then seeded at −6°C and cooled at a rate of 0.3°C/min to −30°C. The straws were then plunged and stored in liquid nitrogen. Embryos frozen in each cryoprotectant were thawed by placing the straws into a 30°C water bath. The embryos were cultured in small dishes in Ham's F-10 medium supplemented with 10% fetal calf serum (FCS). When the embryos were frozen in 0.0, 0.2, 0.4 or 0.8 M sucrose, respectively, in Ham's F-10 the survival rates after culture were 0, 21, 82 or 88%, respectively, in 1.4 M glycerol and 88, 89, 84 or 72%, respectively, in 1.6 M 1, 2-propanediol. Embryos suspended in both cryoprotectants were cooled with 0.2 M sucrose in PBS, and the nonsurgical transfers were performed without removing the cryoprotectant. A pregnancy rate of 61% was obtained when embryos were suspended in 1.6 M 1, 2-propanediol and cooled with 0.2 M sucrose in PBS. However embryos suspended in 1.4 M glycerol and subjected to the same cooling regimen had a lower (P<0.01) developmental rate in vivo following nonsurgical transfer of embryos without the removal of cryoprotectant. These results indicate that 1, 2-propanediol can be used successfully to freeze bovine embryos and that its dilution from bovine embryos can occur in the absence of or at low levels of sucrose.

Nonsurgical collection and transfer of rhesus monkey embryos

Nonsurgical collection and transfer of rhesus monkey embryos

Recovery of bovine oocytes from small vesicular follicles for in vitro maturation and fertilization

Five dairy and four beef breed, mature cows were used as oocyte donors to develop a system of multiple recovery of oocytes for in vitro maturation and fertilization. The animals were alternately treated with either 20 mg of follicle stimulating hormone (FSH) in four equal intramuscular injections or saline at 12 h intervals starting between days 9 and 13 of the oestrous cycle, and the procedure was repeated at three-week intervals for up to four collections. Eighteen collections resulted in the recovery of 124 oocytes from 181 follicles (69%). No serious side-effects were observed. Recovery was equally successful in both breeds and was not reduced in repeat attempts upon the same animal. Treatment with FSH only marginally increased the recovery rate (p less than 0.07) and did not affect the number of follicles aspirated (p greater than 0.05), which varied significantly (p less than 0.05) between cows. From 110 oocytes matured and fertilized in vitro, 70 embryos were recovered after culture in the rabbit oviduct or with trophoblastic vesicles in vitro, of which 30 had cleaved and 5 had progressed to an advanced stage of development. Hormone treatment did not affect zygote development (p greater than 0.05). Four non-surgical transfers of embryos obtained in these studies have resulted in two pregnancies determined ultrasonographically and the birth of a heifer calf. This suggests that the procedure for multiple oocyte recovery is safe and that it can be used successfully for obtaining oocytes for in vitro maturation and fertilization.

Animal biotechnology

Biotechnology has taken two directions in efforts to speed up animal production above the rates achievable by selective breeding. Recombinant DNA methods have been used to engineer protein gene products for direct administration to livestock, as in recombinant growth hormone to stimulate lactation in dairy cows or yield fastergrowing, leaner carcasses in meat animals. Cloned cellulolytic genes have been inserted into ruminal microorganisms with a view to improving ruminant nutrition. The other direction is to use advanced breeding technologies to enhance performance. These include laboratory culture of large numbers of viable embryos for nonsurgical transfer to surrogate mothers, development of methods for sexing sperm and embryos, cloning embryos by nuclear transplantation and gene transfer to create livestock with superior performance traits. In all cases material progress will depend upon a deeper understanding of the underlying physiological and developmental control mechanisms and public confidence that due regard is being paid to animal welfare, and to social and environmental implications.

Induction of twinning in crossbred heifers by ipsilateral frozen embryo transfer

Induction of twinning by means of nonsurgical ipsilateral transfer of two frozen embryos or of demi-embryos was attempted in 297 virgin crossbred dairy heifers that were kept at the following locations: in stables at our breeding station (Group 1), in a mountain field (Group 2) and at a private farm (Group 3). Of these, 187 heifers (63%) were diagnosed pregnant at 25 d after embryo transfer and 134 heifers (45%) were diagnosed pregnant at 60 d after transfer by ultrasonic echography or by palpation per rectum. At calving, 118 heifers produced 46 sets of twins and 72 single calves, for a total of 164 calves sixteen pregnant heifers aborted fetuses between 2 and 7 mo. The pregnancy rate of Group 1 (39%) was lower than that of Group 2 (48%) or Group 3 (50%). Abortion and mortality rates for Group 3 (8% and 8%, respectively) were significantly lower (P<0.05) than for Group 1 (16% and 12%) or Group 2 (13% and 18%). Twin calves had lower birth weights (P<0.05) than singles, but at 270 to 330 d of age, there was no significant difference between the weights of single and twin calves. Dystocia or difficult delivery was not observed heifers producing twins. Retention of the placenta was observed in twin calving heifers, with placenta retention levels the highest in Group 2 (33%) than followed by Group 1 (14%) and Group 3 (10%). When gestation periods were compared between single and twin calves in Groups 1 and 2, the gestation period of single calves was 5.0 to 7.0 d shorter than that of twin calves. No significant difference was observed between the gestation periods of single and twin births in Group 3.

In vitro culture of embryos produced by in vitro maturation and fertilization of bovine follicular oocytes

A previous report from this laboratory dealt with the establishment of pregnancies in the early months of gestation after the non-surgical transfer of cattle embryos derived from the in vitro maturation (IVM) of primary bovine oocytes, their fertilization in vitro (IVF) and their subsequent development to the transferable stage (morula/blastocyst) using an in vivo (sheep oviduct) culture system (Lu et al.,1987). The present report deals with some factors affecting the efficiency of IVF and with the culture in vitro of zygotes to the morula/ blastocyst stage of development. Some embryos were frozen and after thawing transferred by non-surgical procedures to five recipient cattle to obtain information on their capacity to undergo further embryonic development.Primary oocytes, enclosed in cumulus cells, were recovered from vesicular follicles (2-6mm) after their dissection from the ovaries of heifers slaughtered at a local abattoir. The ovaries were brought to the laboratory within one hour of animal slaughter in medium held at 35'C.

Embryo collection and transfer in small ruminants

Recent studies of embryo transfer in small ruminants have yielded laparoscopic and nonsurgical procedures for both embryo collection and transfer. These relatively atraumatic methods for embryo collection produce results that are competitive with surgical methods for collecting uterine stage embryos. Embryos have been collected nonsurgically from sheep, goats, deer, suni antelope and the yellow-backed duiker. Laparoscopic embryo transfer is both rapid and effective when compared with surgical transfer. Although nonsurgical transfers have been achieved in goats, more data are required before this approach can be recommended for widespread application to small ruminants.

Use of embryo transfer to induce twinning in beef cattle: embryo survival rate, gestation length, birth weight and weaning weight of calves.

Experiments were conducted in 1985 and 1986 at the Eastern Ohio Resource Development Center, Belle Valley, to examine the feasibility of using embryo transfer to induce twinning and to examine the influence of twinning on traits of the cow and calf. Embryos were collected from a total of 14 superovulated Angus donors on two dates each in 1985 and 1986 and were transferred to Angus recipients. A total of 124 embryos were transferred to 79 recipients, with 43 (34.7%) calves born alive. Seven of 45 (15.6%) recipients implanted with two embryos produced twins. In no case did both halves of the 15 embryos that were split to produce identical twins and implanted in the same recipient survive to birth. Proportion of calves born alive did not differ among transfer codes 3 (nonsplit embryos from two different donors implanted in separate uterine horns of the same recipient), 6 (nonsplit embryos from one embryo flush implanted in separate uterine horns of the same recipient) and 7 (nonsplit embryos from two different donors implanted in the same uterine horn of one recipient). Surgical transfers tended to result in a higher proportion of embryos surviving to birth (.43 vs .21; P = .16) and a higher twinning rate (.29 vs .04; P = .36) than did nonsurgical transfers. Age of recipient did not influence embryo survival (P = .98) or twinning rate (P = .99). Gestation length was 5 d shorter (P less than .01) for twin calves than for singles. Singles were 9 kg heavier (P less than .01) at birth and 32 kg heavier (P less than .01) at weaning than twins. However, cows raising twins produced 108 kg (51%) more total weaning weight than did cows raising singles.

Nonsurgical embryo transfer in cattle: The effect of clenbuterol on pregnancy rates

Nonsurgical embryo transfer in cattle: The effect of clenbuterol on pregnancy rates

Safety of altrenogest in pregnant mares and on health and development of offspring

Fifty-one light-horse mares were utilized to evaluate the safety of an oral progestin, altrenogest, administered throughout gestation on: gestation length, embryonic and fetal loss, periparturient events, health and development of offspring, and future reproductive capabilities of the mares. Pregnancies were established by inseminating mares with 250 × 10 6 progressively motile spermatozoa from the same stallion every other day throughout estrus or by non-surgical transfer of embryos. Mares were randomly assigned to 1 of 2 treatments upon confirmation of pregnancy on day 20: 1) controls, 2 ml of neobee oil orally per 44.5 kg of body weight; and 2) treated, 2 ml of altrenogest dissolved in neobee oil at a concentration of 2.2 mg/ml orally per 44.5 kg of body weight. Treatments were administered daily from day 20 to 320 of gestation. There were no significant differences between treatment groups for duration of gestation, placental weight, time to placental expulsion and incidence of retained placental membranes. Number of female foals born from altrenogest treated mares (14 of 23) was greater (P<.05) than the number from untreated control mares (4 of 16). Female foals born from altrenogest treated mares had larger clitori (P<.05) than those from control mares. Times to sternal recumbency, standing and nursing were similar for the 2 groups (P>.05). Body weight and height at withers, heart girth circumference and length and width of cannon were measured at time of birth and at 2, 4, 6, 8, 12 and 16 weeks of age. Measurements did not differ (P>05) between treated and control foals for any development parameters. Beginning on day 20 postpartum, mares were teased daily. During estrus, mares were inseminated every other day with 250 × 10 6 motile spermatozoa. Teasing and/or insemination was continued for 2 cycles or until mares were 35 days pregnant. The number of mares pregnant after 1 cycle and after 2 cycles of insemination was similar (P>.05) for treated and control mares. Nineteen of 21 treated mares and 15 of 16 control mares were pregnant after 2 cycles of insemination. Number of cycles per pregnancy was similar (P>.05) for treated and control mares (1.37 vs 1.13) as was number of days mares exhibited estrus (6.30 vs 6.13). Number of inseminations per cycle did not differ (P>.05) between treated and control mares (2.92 vs 3.00). In summary, there was no effect of treatment with altrenogest from day 20 to 320 of gestation on periparturient events, viability and growth of offspring and subsequent reproductive performance of mares.

Induction of twinning in dairy or crossbred heifers by ipsilateral frozen embryo transfer.

Induction of twinning by ipsilateral nonsurgical transfer of frozen two or demi-embryos was attempted in 129 virgin dairy or crossbred heifers that had been kept under a stable in our station (group-1), mountain field (group-2) and private farm (group-3) conditions. Ninety seven heifers (75%) were diagnosed pregnant by rectal palpation at 35 to 60 days of gestation; 86 heifers produced 37 sets of twins and 49 single calves. Pregnancy rate of group-1 was lower compared to those of groups-2 and -3 (63%, 88%, 78%, respectively). Abortion and mortality rates of group-3 were significantly lower (P less than 0.05) than those of groups-1 and -2 (8% and 6%, 12% and 16%, 18% and 24%). Twin calves had a lower birth weight (P less than 0.05) than singles. But there was no significant difference in weight between singles and twin calves at 270-330 days of age. Dystocia and difficult delivery were not observed in females producing twins. The incidence of retained placentas in twin calving cows (17%) was higher than that of single calving cows (2%). The interval to conceive postpartum was longer in twin calving cows (95 +/- 41 days) than in single calving cows (87 +/- 26 days). Gestation length was 5.0 days shorter for twin calving cows (P less than 0.05).

The culture of bovine oocytes to obtain developmentally competent embryos.

Bovine cumulus-oocyte complexes (COC) (n = 4230) were used in this study to assess the effects of culture method, hormonal supplementation, and cumulus cell concentration on maturation, fertilization and development of resulting embryos. Five treatments were evaluated. 1) 10 COC/50-microliter drops under oil in TCM 199 supplemented with 10% heat-treated fetal calf serum, follicle-stimulating hormone (0.5 microgram/ml), luteinizing hormone (5 micrograms/ml), and estradiol-17 beta (1 microgram/ml); 2) as in 1 without hormones; 3) as in 1 but in 3 ml TCM-199 in petri dishes without paraffin oil; 4) as in 2 but only 1 COC/50-microliter drop; and 5) as in 1 but with denuded oocytes. After 24 h maturation, the frequencies of oocytes reaching metaphase II were 98, 84, 92, 93, and 87%, respectively, for the five treatments. In the same order, percentages of normal fertilization were 73, 70, 62, 81, and 62%, and the frequencies of embryos containing two or more blastomeres at 65 h postinsemination were 69, 82, 66, 51, and 43%. The same five treatments were used in a second study in which 3,199 oocytes were fertilized, allowed to cleave in vitro to the 2- to 3-cell stage (42 h postinsemination), and transferred to oviducts of sheep (one treatment/oviduct) for 4 days. The frequencies of morulae or blastocytes obtained were 28, 18, 23, 24, and 11% for the five treatments, respectively. After nonsurgical transfer to bovine recipients (n = 8) using fresh or frozen-thawed embryos, three pregnancies past 50 days were obtained. Only one went to term with the birth of a live heifer calf.

Embryo transfer: its uses and recent developments.

The technique of embryo transfer is now routinely employed in several species and with several objectives. In laboratory animals the technique is used mainly in investigations of reproductive biology, whereas in human beings it is used to overcome specific forms of impaired fertility. Because embryo transfer combined with superovulation of the donor can significantly increase the female reproductive rate its greatest application to date has been in farm animals, in which it is widely used in both research and commercial production. Within the past few years there have been many advances in the techniques used in farm animals, particularly in the area of embryo manipulation. The supply of embryos can now be increased by repeated superovulation and by embryo bisection and there has been significant progress in in vitro fertilisation technology. Deep freeze (-196 degrees C) storage of embryos is now routine and may be combined with direct one-step thawing and removal of cryoprotectant. This technique allows the routine non-surgical transfer of embryos in the field. There is also evidence that a routine non-traumatic procedure for sexing embryos may soon be developed. Identical multiplets or clones have been produced by the microdissection of embryos and more recently by the transfer of nuclei from embryos into unfertilized oocytes. Transgenic animals have been produced by the microinjection of recombinant DNA into one of the pronuclei of single-cell unfertilized ova.

Development of Bovine Follicular Oocytes Matured and Fertilized In Vitro by Co-culture with Cumulus Cells and subsequent Pregnancies by Non-surgical Transfer

Development of Bovine Follicular Oocytes Matured and Fertilized In Vitro by Co-culture with Cumulus Cells and subsequent Pregnancies by Non-surgical Transfer

Nonsurgical transfer and the survival of frozen-thawed bovine embryos supplemented with raffinose

Nonsurgical transfer and the survival of frozen-thawed bovine embryos supplemented with raffinose

Pregnancy resulting from cattle oocytes matured and fertilized in vitro.

Follicular oocytes (n = 81) collected from cattle at a local slaughterhouse were matured and fertilized in vitro. Of 27 ova 19 (70%) were penetrated by spermatozoa and 40/54 (74%) inseminated ova transferred surgically to the oviducts of a synchronized heifer were recovered by non-surgical flushing of the uterine horns 6 days later. Of the 40 ova 15 (38%) were at the morula, early blastocyst or diminutive morula stages. Culture in vitro sustained further development of all embryos and 9 were expanding or expanded blastocysts. One pregnancy resulted from non-surgical transfer of 2 blastocysts. The results demonstrate that immature oocytes from cattle can be matured and fertilized in vitro, subsequently develop to the blastocyst stage, and develop into a normal pregnancy after non-surgical transfer.

Effects of slitting the zona pellucida and its subsequent sealing on freeze-thaw survival of Day 7 bovine embryos

The effects of slitting the zona pellucida and its subsequent sealing by either embedding in agar or surrounding with an additional zona pellucida on the development of frozen/thawed Day 7 bovine embryos were investigated in vitro and in vivo. A total of 225 embryos was frozen and thawed rapidly as controls (Group 1), after slitting the zona pellucida (Group 2), after slitting and subsequent sealing of the zona pellucida with agar (Group 3), or after slitting the zona pellucida (Grothen transferring the embryo into an additional zona pellucida (Group 4). The survival rate (embryos classified morphologically as excellent, good, or poor) was 95.1, 95.4, 92.2, and 94.3% for Groupsl, 2, 3, and 4, respectively. Culture of 145 embryos in vitro for 60 h revealed that 57.1, 59.5, 47.4, and 57.1% developed to hatching and hatched blastocysts in Groups 1, 2, 3, and 4, respectively. Within Group 3, however, a significantly (P < 0.05) lower percentage of the embryos continued to develop when the agar was not removed after thawing (31.8%) compared with embryos from which the agar had been removed (68.8%). After nonsurgical transfer of 78 embryos, the pregnancy rate was significantly (P < 0.05) lower (8.3%) with embryos of Group 3 compared with controls (61.5%) or embryos of Group 2 (42.9%). No significant difference existed between controls and embryos of Group 2. We conclude that an intact zona pellucida prior to rapid freezing is not essential for the survival of Day 7 bovine embryos.

Successful transfer of the embryos of Przewalski's horses (Equus przewalskii) and Grant's zebra (E. burchelli) to domestic mares (E. caballus).

Blastocysts were collected non-surgically from 2 Przewalski's horse and 2 Grant's zebra mares and transferred extra-specifically to domestic horse and donkey recipients. Nine Przewalski's horse embryos were transferred surgically, and 2 non-surgically, to domestic Welsh-type pony mares. After surgical transfer, 7 (77.8%) pregnancies were established and 4 foals were born. Twelve Grant's zebra embryos were transferred surgically to 5 pony and 7 domestic donkey recipients respectively and 1 non-surgically to a donkey; 3 (60%) zebra-in-horse pregnancies were established and 2 went to term. Only 2 (28.6%) zebra-in-donkey pregnancies were established but neither went to term, although one zebra foal was aborted alive at Day 292 but failed to survive. No pregnancies resulted from the non-surgical transfers. Measurement of chorionic gonadotrophin concentrations and parental-specific lymphocytotoxic antibodies in the serum of the recipient animals indicated a pronounced maternal immunological response to the extra-specific embryo, but this could not be correlated with success or failure of pregnancy. The results indicate that extra-specific embryo transfer may be a useful aid to breeding exotic equids in captivity.

Non-surgical recovery and transfer of equine embryos

Non-surgical recovery and transfer of equine embryos

Successful non-surgical transfer of equine embryos to post-partum lactating mares

Successful non-surgical transfer of equine embryos to post-partum lactating mares