R.P. Elsden

Superovulating cows with follicle stimulating hormone and pregnant mare's serum gonadotrophin

A total of 511 embryos was recovered non-surgically from nearly 100 superovulated or untreated donors. Superovulation with FSH-LH resulted in more corpora lutea, recovered ova, and pregnancies (P<.01) than superovulation with PMSG. No differences were observed in numbers of ovulations, embryos recovered, or pregnancies per donor when prostaglandin F2α was given to donors 2 versus 3 days following gonadotrophin treatment. Pregnancy rates of 12, 31, 58, and 63% were obtained from groups of embryos classified morphologically as poor, fair, good, and excellent (P .05) between pregnancy rates when retarded embryos from untreated donors (12%) were compared to retarded embryos from superovulated donors (22%). However, a higher proportion of morphologically normal embryos from untreated donors developed into fetuses (71%) than did morphologically normal embryos from superovulated donors (59%, P<.05).

Non-surgical recovery of bovine eggs

Abstract An improved, non-surgical technique for recovering bovine eggs is described using modified Foley catheters. Egg recovery rates per attempt were, 36 51 (71%), for normal, unsuperovulated donors and 4 38 (11%), for unsuperovulated donors with known fertility problems. For superovulated donors, eggs were collected in 24 26 attempts (92%) averaging 6.9 per recovery.

Pregnancy rates with bisected bovine embryos.

A method for producing identical twin calves is described in which 5.5 to 7.5 day bovine embryos were bisected using a fine microsurgical blade. The resulting demi-embryos were transferred surgically or nonsurgically to the uterine horn ipsilateral to the corpus luteum of synchronous recipients (+/- 2 days), one demi-embryo per recipient. The mean pregnancy rate per demi-embryo was 48% (157/330), and per whole embryo split, it was 95% (157/165). Pregnancy rate was dependent on the stage of embryonic development. The pregnancy rate from bisected early morulae, 16% (7/44), was lower than that from bisected early blastocysts, 60% (58/96) P<0.01. The rate of identical twin production per embryo split reflected these pregnancy rates. Bisected blastocysts resulted in more identical twin sets, 39% (24/62), than early morulae, 9% (2/22) P<0.01. When used with normal embryo transfer procedures, this technique provides a method for routine production of identical twin calves. Furthermore, more calves are produced per two demi-embryos than per whole embryo.

A new procedure for the cryopreservation of equine embryos.

Early equine blastocysts and blastocysts were collected nonsurgically at six days post-ovulation. Thirty-two embryos were randomly assigned to a 2x2 factorial design. Factors were: 1) 0.5-ml straws or 1-ml glass ampules; and 2) plunging into liquid nitrogen (IN(2)) at -33 C or -38 C. Cryoprotectant, 10% glycerol in PBS plus 5% fetal calf serum (FCS) was added in two steps, 5% then 10%. Embryos were cooled at 4 C/min to -6 C and then seeded, 0.3 C/min to -30 or -35 C and 0.1 C/min to -33 or -38 C. Samples were thawed in 37 C water and glycerol removed in six steps, 10 min per step. Embryo quality and stage of development were evaluated prior to freezing, immediately post-thaw and after 24 h culture in Hams F10 with 5% FCS. The mean post-thaw quality of embryos plunged at -33 C was superior (P<0.05) to that of embryos plunged at -38 C (2.0 vs 2.9). Embryos frozen in ampules and plunged at -38 C were of poorer quality (P<0.05) than those frozen in ampules and plunged at -33 C or frozen in straws and plunged at -33 C. After 24 h of culture, more embryos developed if frozen in straws compared to ampules, and plunging at -33 C resulted in higher quality embryos than plunging at -38 C. In Experiment 2, 23 embryos were packaged in straws and plunged at -33 C as described in Experiment 1. Six of the 23 surgically transferred frozen embryos were degenerate at thawing and the remaining 17 surgically transferred were via flank incision. Pregnancy rate at 50 days post-ovulation was 53% (nine of 17). Early blastocysts resulted in a higher (P<0.05) pregnancy rate (8 10 , 80%) than expanded blastocysts (1 7 , 14%).

Field experiments with frozen-thawed bovine embryos transferred nonsurgically

Abstract Two hundred and twenty 6- to 8-day embryos graded good to excellent were frozen in modified Dulbeccos phosphate-buffered saline + 20% fetal calf serum. Three cryoprotectants, added in three steps, were compared: 1.5 M ethylene glycol (EG), .625 M dimethyl sulfoxide + .625 M glycerol (D+G), and 1.33 M glycerol (G). Following cooling at 1 C/min to −7 C, ampules were seeded followed by one of three cooling methods prior to plunging into liquid nitrogen: 1) 0.3 C/min to −35 C, then holding for 30 minutes, 2) 0.3 C/min to −38 C and 3) 0.3 C/min to −35 C, then 0.1 C/min to −38 C. Thawing at 25 C or 37 C was followed by a 6-step dilution of cryoprotectant. One hundred and fifty-three embryos were then transferred nonsurgically into synchronized recipients. At the same time, 35 fresh embryos were transferred to recipients as controls. No significant differences were found among cryoprotectants. A significantly (P 26 35 ) resulted from nonsurgical transfer of unfrozen embryos. The optimum treatment combination was glycerol as cryoprotectant (1.33 M), cooling at 0.3 C/min to −35 C, then 0.1 C/min to −38 C, plunging into liquid nitrogen and thawing at 37 C. With this combination of treatments, a pregnancy rate greater than 50% (based on embryos frozen) was obtained following nonsurgical transfer.

Estimation of embryonic losses in bovine embryo transfer recipients from progesterone profiles and returns to estrus

Abstract Progesterone profiles for 212 embryo transfer recipients were determined by obtaining blood samples every 3.5 days. Observations for behavioral estrus plus progesterone profiles were used to determine when pregnancies were lost in nonpregnant recipients. Of 19 recipients receiving embryos of poor morphological quality, only five maintained pregnancy beyond the length of a normal estrous cycle, and one of these pregnancies was lost by 2 months of gestation. Of 193 recipients receiving morphologically fair to excellent embryos, 130, or 67.4%, were pregnant at 2 months. Pregnancy in eight of these recipients (4.1%) did not progress beyond day 16 from the pretransfer estrus because of short or inadequate luteal phases. Pregnancy did not progress beyond day 24 for another 43 recipients (22.3%), that returned to estrus within a normal estrous cycle or had progesterone profiles indicative of a normal estrous cycle and apparent silent or missed estrus. The remaining 12 recipients (6.3%) had progesterone profiles which dropped to less than 2 ng/ml of blood serum between days 31 and 59, and probably represent embryonic death within this time frame. Thus, 12 of the 142 recipients (8.5%) estimated to be pregnant on day 25 were no longer pregnant at 2 months. An additional study of returns to estrus of 910 recipients resulted in the following observations: 54.7% were pregnant at 2 months; 1.5% (n=14) returned within 2–16.5 days; 24.4% (n=222), between 17–24 days; 2.6% (n=24), between 24.5 and 34 days; 9.2% (n=84), between 34.5 and 46.5 days; and 7.5% (n=68), after 47 days. Undoubtedly there were some silent or missed estruses, but these returns in general corroborate information obtained from progesterone profiles. Recipients receiving morphologically poor embryos not only had lower pregnancy rates by day 24, but in addition, those pregnant on day 24 had a higher incidence of embryonic death compared to those receiving morphologically normal embryos. We conclude that 73.6% of recipients receiving morphologically normal embryos remained pregnant on day 24, and 67.4% were still pregnant at 2 months. From a related study, we would expect 63.6% to produce calves at term, and 57.9% to have live 1-month-old calves. The causes of many of these losses remain unknown; additional research may lead to methods of reducing or circumventing some of this wastage.

Embryo transfer in fertile and infertile cows

Abstract Pregnancy rates were compared between fertile and infertile donors in an embryo transfer program. Non-surgical embryo transfer techniques were utilized for examination of uterine contents and collection of ova. Recovered embryos were transferred surgically into suitable recipients. Three groups of donors were included in this investigation: a) fertile; b) infertile, due to known causes (diagnosed); c) infertile, due to unknown causes (undiagnosed). There were 11.3, 6.6 and 8.0 corpora lutea; 6.8, 1.2 and 1.0 fertilized ova; 3.6, 0.8 and 0.4 pregnancies per superovulation for the fertile, diagnosed and undiagnosed groups, respectively. In 23 months, unsuperovulated, fertile, diagnosed and undiagnosed donors yielded 17, 15 and 9 fertilized ova which resulted in 15, 13, and 3 pregnancies, respectively. Donors treated for uterine infections, adhesions or cystic ovaries prior to superovulation responded with 10.1, 7.6 and 4.1 corpora lutea; 1.9, 4.0 and 0.3 fertilized ova; 1.1, 3.3 and 0.2 pregnancies per superovulation, respectively. Relatively few viable embryos were recovered from donors with chronic cystic ovaries or from infertile cows of unknown etiology. Infertile donors, when compared to fertile donors, were unproductive when used for embryo transfer.

Development and viability of frozen-thawed cattle embryos

Embryos recovered 7 to 8 days after estrus were frozen from -7 to -30 degrees C at 0.3 degrees C/min, from -30 to -33 degrees C at 0.1 degrees C/min, and then plunged into liquid nitrogen. They were thawed in a 25 degrees C waterbath. In a preliminary study, 15 of 18 embryos continued to develop during the 24-hour culture post-thaw in either Hams F-10 or modified Dulbeccos phosphate buffered saline (PBS). In the main study, 5 of 20 embryos developed to 60-day pregnancies when embryos were transferred within 5 hours after thawing. The incidence of extended estrous cycles (pregnancy or presumed embryonic mortality) was 10 of 14, when the zona pellucida was intact after thawing, and 0 of 6, when it was ruptured or absent (P<.05). Embryos cultured in PBS tended to develop more readily than those in Hams F-10 (15 of 20 vs 9 of 20, respectively, P reverse similar.1). Quality of the embryos, at recovery from the donor and after thawing, affected development in culture (19 of 27 embryos excellent at recovery developed vs 5 of 13 poor to very good, P reverse similar.1; 23 of 33 embryos good to excellent after thawing developed vs 1 of 7 poor, P<.05). The proportion of pyknotic nuclei in embryos which were cultured ranged from 18 to 100%. The pregnancy rate from embryos which were cultured was low (2 of 20). Thirty percent of frozen and thawed embryos had damaged zonae pellucidae. The study showed that: the pregnancy rate from frozen embryos was approximately half that achieved with unfrozen embryos; culturing embryos for 24 hours before transfer was not beneficial; the PBS culture system appears to be the system of choice for assessing embryo viability in vitro .

Intergeneric embryo transfer between water buffalo and domestic cattle

Abstract The feasibility of reciprocal embryo transfer between water buffalo ( Bubalus bubalis ) and domestic cattle ( Bos taurus ) was studied. Eight mature water buffalo females were superovulated and bred naturally to a male water buffalo. Sixteen water buffalo embryos were recovered nonsurgically using routine bovine embryo transfer procedures. No pregnancies resulted after transfer of thirteen water buffalo embryos to synchronized Holstein heifers. Physiological age of the water buffalo embryos was ahead of chronological age when compared to bovine embryo development. Two cattle embryos were transferred nonsurgically to two synchronized water buffalo recipients. One recipient was diagnosed pregnant; however, she subsequently aborted between 2.5 and 3.0 months of gestation. The significance of successful intergeneric embryo transfer between cattle and water buffaloes would be the provision of a model for the preservation of endangered genera and species for which common recipients are readily available. The reproductive anatomy as well as the reproductive physiology of the estrous cycle of the donor and the recipient species must be reasonably similar. Minor endocrinological incompatibilities might be circumvented by transfer of alien embryos to mated recipients. Problems of immunological incompatibilities might be minimized by modifying the immune response of the recipient and/or the antigenicity of the embryo by microsurgical manipulation.

Non-sugrical embryo transfer in the cow

Abstract Embryos were recovered from superovulated heifers and transferred non-surgically through the cervix of heifers via an ureteral catheter. The ureteral catheter was passed into the anterior portion of the uterine horn ipsilateral to the corpus luteum through a series of concentric stainless steel tubes. One morula or blastocyst was transferred to each of 95 heifers. Eighteen heifers (19%) were pregnant 55 to 60 days later. The pregnancy rate was not significantly influenced by treatment of the vagina with antibiotics before transfer; whether a morula or blastocyst was transferred; or whether ovulation occurred from the left or right ovary. There were no significant differences among the three technicians. Fifty of the 77 recipients that were not pregnant at 60 days of gestation were observed in estrus after 18–23.5 or 37–44.5 days, representing normal or double the normal estrous cycle lengths. However, 11 showed a shortened return to estrus (9–26 days), and 16 had a delayed return to estrus (25–33 or 48–55 days). We conclude that this technique results in premature luteolysis in some recipients and probably in more early embryonic death than normal.