Factors Affecting Pregnancy Rate Per Embryo Transfer of In Vivo Produced Embryos in Recipient Holstein Heifers

Despite the rapid expansion of in vitro embryo production (IVP) technology for genetic improvement in the cattle industry in the last decades, pregnancy rates by the transfer of IVP embryos are still lower than those derived from in vivo-produced embryos. The objective of this study was to analyse factors affecting pregnancy rates after the transfer of IVP Japanese Black cattle embryos under farm conditions. Holstein heifers (n = 4,475) and cows (n = 8,541) were selected as recipients. Most cows (80%) were managed in tie-stall barns and most heifers (80%) were managed in pens. Embryo transfers were performed for 9 years, from 2004 to 2012. The embryos were produced from oocytes derived from a local abattoir and semen from 14 proven bulls by the Livestock Improvement Association of Japan (Hamano and Kuwayama 1993 Theriogelogy 39, 703–712). The fresh IVP embryos (quality; IETS code 1) that reached the blastocyst stage after 7 to 8 days (insemination = Day 0) were transported by an airplane (2 h) and subsequently by a car (1.5 h). Embryos were non-surgically transferred to each recipient on Day 7 to 9 of their natural oestrous cycle on farms. Pregnancy was diagnosed on Day 40 to 60 after oestrus. Pregnancy results were statistically analysed using the GLM procedures of SAS. The following variables were included in the model: recipient parity (0, 1, 2, or 3), day (7, 8, or 9) of the oestrous cycle at the time of embryo transfer, oestrus behaviour (increased activity observed by farmers), presence of mucus at oestrus, presence of blood after oestrus, and year (1, 2, 3, 4, 5, 6, 7, 8, or 9) and season (April–June as spring, July–September as summer, October–December as fall, or January–March as winter) of embryo transfer. The Bonferroni correction was used to counteract the problem of multiple comparisons. Heifers had significantly higher pregnancy rates than cows (51.0% v. 37.9%, respectively; P < 0.01), and first parity cows had higher pregnancy rates than third parity cows (42.9% v. 35.7%, respectively; P < 0.01). Pregnancy rates were significantly higher in recipients that received an embryo transfer on Day 8 of their oestrous cycle, than on Day 7 (46.6% v. 42.4%, respectively; P < 0.01). Recipients without oestrus behaviour had higher pregnancy rates than those with oestrus behaviour (46.3% v. 43.4%, respectively; P < 0.01). The presence of mucus and/or blood after oestrus and the season of transfer were not found to significantly affect pregnancy rates. The results of this study indicated that performing IVP embryo transfers on Day 8 of a recipient’s oestrous cycle will improve the pregnancy rate, season does not have an effect on pregnancy rate, and the detection of oestrus by monitoring increased activity is not always reliable and instead should be determined by multiple symptoms on farm conditions.

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39 FACTORS AFFECTING PREGNANCY RATE FOLLOWING TRANSFER OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS IN DOGS

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab39 ◽

2009 ◽

Vol 21 (1) ◽

pp. 119

Author(s):

M. J. Kim ◽

H. J. Oh ◽

J. E. Park ◽

J. T. Kang ◽

S. G. Hong ◽

...

Keyword(s):

Somatic Cell ◽

Pregnancy Rate ◽

Embryo Transfer ◽

Serum Progesterone ◽

Factors Affecting ◽

Oocyte Donor ◽

Surgical History ◽

History Of ◽

Analysis System

There have been attempts to find out factors influencing pregnancy rate after transfer of somatic cell nuclear transferred (SCNT) couplets in several species. The purpose of this study was to investigate the effects of parity, surgical history, and synchronization state between oocytes donor and recipient on the pregnancy rate following transfer of SCNT embryos in dogs. A total of 25 mixed-breed female dogs, aged 1 to 5 years old, weighing 20 to 35 kg were used in this study. In vivo-matured oocytes, obtained by flushing uterine tubes of donor dogs approximately 72 h after ovulation, were used for SCNT. Ovulation was determined by daily serum progesterone concentration. Reconstructed embryos (range = 7–36, average = 18.0 embryos) with normal morphology were surgically transferred into the ampullary portion of the uterine tube of recipient dogs. Pregnancy was determined at least 26 days after transfer by untrasonography. In experiment 1, recipient females were divided into 2 groups: nulliparous (7 bitches) and multiparous (17 bitches) groups. In experiment 2, recipient females were divided into 2 groups by surgical history: operated (10 bitches) and non-operated (14 bitches) groups. Operated group included those with a history of 1 laparotomy, either flushing of uterine tubes or SCNT embryo transfer. Non-operated group included those without history of any surgery. In experiment 3, recipient females were divided into 2 groups: synchronous (13 bitches) and asynchronous (7 bitches) groups. Synchronous was defined that the oocyte donor dog and the recipient dog ovulate at the same day. Asynchronous was defined that the oocyte donor dog ovulates 1 day earlier than the recipient. Data were analyzed using the Statistical Analysis System program (SAS Institute, version 9.1, Cary, NC, USA). In experiment 1, pregnancy rate was significantly higher in the multiparous group than the nulliparous group (23.5 v. 14.2%, P < 0.05). In experiment 2, surgical history did not influence the pregnancy rate between operated and non-operated groups (20.0 and 21.4%, respectively). In experiment 3, pregnancy rate was significantly higher in the asynchronous group than the synchronous group (42.9 v. 15.4%, P < 0.05). In conclusion, multiparous bitches improved the efficiency of SCNT embryo transfer. A higher pregnancy rate resulted when the oocyte donor dog’s ovulation time was 1 day earlier than that of the recipient dog. Other factors should be investigated for further improvement of the SCNT embryo transfer efficiency. This study was financially supported by the Korean MEST, through KOSEF (grant # M10625030005-08N250300510) and the BK21 program for Veterinary Science, and SNU foundation (Benefactor; RNL BIO).

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Factors affecting pregnancy rate following nonsurgical embryo transfer in buffalo (Bubalus bubalis) : A retrospective study

Theriogenology ◽

10.1016/s0093-691x(99)00105-3 ◽

1999 ◽

Vol 52 (1) ◽

pp. 1-10 ◽

Cited By ~ 7

Author(s):

A.K. Misra ◽

M. Mutha Rao ◽

R. Kasiraj ◽

N.S. Ranga Reddy ◽

H.C. Pant

Keyword(s):

Retrospective Study ◽

Pregnancy Rate ◽

Embryo Transfer ◽

Bubalus Bubalis ◽

Factors Affecting

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Factors affecting pregnancy rate in embryo transfer recipients with reference to plasma progesterone

Theriogenology ◽

10.1016/0093-691x(89)90646-8 ◽

1989 ◽

Vol 31 (1) ◽

pp. 238 ◽

Cited By ~ 4

Author(s):

A.J. Payas ◽

P.J. Broadbent ◽

D.F. Dolman ◽

W.B. Christie

Keyword(s):

Pregnancy Rate ◽

Embryo Transfer ◽

Plasma Progesterone ◽

Factors Affecting

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Studies on factors affecting superovulation and embryo transfer in Hungarian Merino ewes

Acta Veterinaria Hungarica ◽

10.1556/004.49.2001.4.7 ◽

2001 ◽

Vol 49 (4) ◽

pp. 431-441 ◽

Cited By ~ 2

Author(s):

S. Cseh ◽

L. Solti

Keyword(s):

Developmental Stage ◽

Pregnancy Rate ◽

Embryo Transfer ◽

Ovarian Stimulation ◽

Corpora Lutea ◽

Treatment Protocols ◽

Factors Affecting ◽

Factors Influencing ◽

Significant Difference ◽

One Year

The objectives of this study were (a) to assess the ovulatory response and embryo production of Hungarian Merino ewes after superovulation, (b) to investigate the factors influencing the efficiency of embryo transfer (ET) in Hungarian Merino ewes, (c) to compare the results of two ovarian stimulation protocols (PMSG and PMSG+FSH treatment) in Hungarian Merino ewes, and (d) to study how superovulation, laparoscopic insemination and surgical embryo retrieval (ER) affect the subsequent reproduction of Hungarian Merino donor females after an ET programme. There was no significant difference between the ovarian stimulation protocols in the ratio of donor ewes responding to superovulation nor in the average number of corpora lutea. However, the number of transferable embryos recovered per donor ewe was higher in the PMSG+FSH group. The proportion of transferable embryos, unfertilised oocytes and degenerated embryos did not differ between the treatment protocols. The total pregnancy rate was 53.4% (179/335). Neither the developmental stage of the embryo nor the number of transferred embryos affect the implantation of embryos. However, the increased number of transferred embryos positively influenced the pregnancy rate. No difference was found in the pregnancy rate between synchronised and nonsynchronised groups of recipients. Thirty-six out of 45 donor ewes (80%) became pregnant within one year after the ET programme, indicating that ovarian stimulation and surgical ER did not affect adversely their reproduction.

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204 RELATIONSHIP BETWEEN RESPIRATORY ACTIVITY AND THE PREGNANCY RATE OF BISECTED BOVINE EMBRYOS IN VIVO

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab204 ◽

2007 ◽

Vol 19 (1) ◽

pp. 219 ◽

Cited By ~ 2

Author(s):

S. Moriyasu ◽

H. Hirayama ◽

K. Sawai ◽

S. Kageyama ◽

S. Aoyagi ◽

...

Keyword(s):

Oxygen Consumption ◽

Pregnancy Rate ◽

Embryo Transfer ◽

Respiratory Activity ◽

Pregnancy Rates ◽

Bovine Embryos ◽

Chi Square ◽

Important Indicator ◽

Consumption Rates

Oxygen consumption is an important indicator of the metabolic activity of living cells, which may provide valuable information for evaluating embryo quality. We have found that the bovine embryos with high oxygen consumption possess stronger potential for further development. However, the relationship between respiratory activity and the pregnancy rate of embryos is still unclear. In this study, we investigated the respiration rates of bisected bovine embryos and the pregnancy rates of demi-embryos after embryo transfer. Compact morula-stage embryos were bisected evenly by micro glass needle. One hundred bisected embryos were incubated for 24 h in embryo culture medium (IVD101; Research Institute for the Functional Peptides, Yamagata, Japan) at 39�C under 5% CO2, 5% O2, 90% N2. After the incubation, demi-embryos were classified into 2 groups: blastocoel-formed (BC) and blastocoel-not-formed (CM) embryos. Oxygen consumption rates of demi-embryos were measured by scanning electrochemical microscopy (SECM; Hokuto Denko Corporation, Tokyo, Japan). Within 3 h after the measurement, 80 demi-embryos were transferred into recipient cows (one demi-embryo/one recipient) at 7–8 days after estrus. Recipient cows were diagnosed for pregnancy by ultrasonography approximately 40 days after estrus. Statistical difference was analyzed by Tukey's post-hoc test and chi-square test. A total of 27 recipient cows became pregnant; the pregnancy rates for cows with CM and BC demi-embryos were 40.6% (13/32) and 29.2% (14/48), respectively. Mean oxygen consumption rates (� 10-14 mol s-1) in pregnant and non-pregnant cows were 0.47 and 0.39 for CM demi-embryos and 0.63 and 0.52 for BC demi-embryos, respectively. Retrospective analysis showed that the respiratory activity of demi-embryos in the pregnant group was higher than those in the non-pregnant group. In particular, the pregnancy rates for demi-embryos with respiratory activity higher than 0.35 in CM and 0.40 in BC groups were 52.0% (13/25) and 35.9% (14/39), respectively. On the other hand, cows with demi-embryos having an oxygen consumption rate under 0.35 in CM (n = 7) and 0.40 in BC (n = 9) groups did not become pregnant. These results demonstrated that bovine demi-embryos with higher respiratory activity showed a high pregnancy rate after embryo transfer. It is generally known that the pregnancy rate after the transfer of bisected embryos is lower than that of whole embryos. The measurement of oxygen consumption by SECM procedures is a useful tool to assess the quality of pre-implantation embryos and may contribute to the improvement of the success rate for bisected embryo transfer.

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37 EFFECT OF EMBRYO STAGE ON PREGNANCY RATE FOLLOWING DIRECT TRANSFER OF BOVINE EMBRYOS FROZEN IN ETHYLENE GLYCOL

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab37 ◽

2012 ◽

Vol 24 (1) ◽

pp. 131 ◽

Cited By ~ 3

Author(s):

J. F. Hasler

Keyword(s):

Ethylene Glycol ◽

Pregnancy Rate ◽

Embryo Transfer ◽

The United States ◽

Pregnancy Rates ◽

Bovine Embryos ◽

Embryo Freezing ◽

Percentage Points ◽

Embryo Stage

Annually, more than 400 000 in vivo-recovered bovine embryos are officially reported by members of the Canadian and American Embryo Transfer Associations. Between 65 and 70% of these embryos are cryopreserved and more than 95% are frozen in ethylene glycol (EG). Statistics on factors affecting embryo freezing are difficult to obtain because many cattle breeders/farmers no longer report pregnancy rates back to embryo transfer (ET) practitioners. Concerns are often expressed as to the optimal stage at which to freeze bovine in vivo-derived embryos. This is a retrospective analysis of results from 5 commercial ET programs (1 in the United States, 3 in Canada and 1 in the Netherlands) for which pregnancy data relative to embryo stage at freezing were made available. Embryos representing 4 stages of development, as defined by the IETS (4 = late morula, 5 = early blastocyst, 6 = mid blastocyst and 7 = expanded blastocyst) are included in the data. The number of embryos thawed and transferred ranged from 3954 to 24 827 for the 5 programs, with a total of 72 828. Embryos were frozen in either 1.5 M EG or 1.5 M EG + 0.1 M sucrose and exposure time to cryoprotectant before cooling ranged from 4 to 40 min. Pregnancy rates are shown in Table 1. Although the pregnancy rate for stage 6 embryos was only 2.6 and 3.2 percentage points lower than stages 4 and 5, respectively, these differences were highly significant and pregnancy rates for stage 6 embryos were lower than those for stages 4 and 5 in 4 of the 5 ET programs. The small decreased survival of stage 6 embryos is probably only moderately important in a commercial context. However, the pregnancy rate of stage 7 embryos was lower than all other stages for the combined dataset as well as in all 5 ET programs, with the difference between stages 5 and 7 ranging from 6.5 to 16.4 percentage points. Clearly, stage 7 embryos survive freezing at a significantly lower rate than stages 4, 5 and 6 and neither time of exposure to EG nor inclusion of sucrose in the freezing medium provided an obvious improvement. Although bovine ET practitioners routinely attempt to collect embryos on day 7 post-oestrus, recovery of stage 7 embryos cannot always be avoided. Further investigation into factors contributing to the decreased survival of stage 7 embryos is warranted. Table 1.Effect of embryo stage on pregnancy rate of bovine embryos frozen in EG

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363 FACTORS INFLUENCING PREGNANCY RATES FOLLOWING TRANSFER OF BOVINE IN VIVO EMBRYOS BIOPSIED FOR SEX DETERMINATION

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab363 ◽

2007 ◽

Vol 19 (1) ◽

pp. 297

Author(s):

S. Li ◽

W. Yu ◽

J. Fu ◽

Y. Bai ◽

F. Jin ◽

...

Keyword(s):

Pregnancy Rate ◽

Embryo Transfer ◽

Pregnancy Rates ◽

Chi Square ◽

Embryo Survival ◽

Chi Square Test ◽

First Time ◽

Fresh Embryos

Data collected from commercial embryo transfer programs in 63 farms in China during June 2002 to December 2005 was analyzed to examine the effects of various factors (biopsy, freezing, sample size, embryo development and quality, in vitro culture, and recipient quality) on pregnancy rates of in vivo-biopsied embryos. Embryos were flushed from superovulated dairy cattle and subjected to a biopsy for sexing determination using protocols and sexing kits supplied by AB Technology Ltd. Fresh embryos were implanted on the same day or frozen with AG freeze medium (AB Technology Ltd., Pullman, WA, USA) for later transfer. Recipients were synchronized with CIDA + PG protocols. Embryos were cultured in 6-well dishes containing 1.3 mL of holding medium (AB Technology Ltd.) in each well at room temperature (20–25�C) for examination of embryo survival in vitro. The chi-square test was used in statistic analysis. The implantation of fresh embryos after biopsy did not affect pregnancy rates (49.6%, 257/518) compared to that of non-biopsied fresh and frozen–thawed embryo groups (52.9%, 47/140 and 46.6%, 177/380, respectively). However, for biopsied embryos subjected to frozen and thawed procedures before implantation, particularly for those subjected to the removal of a larger biopsy, a reduced pregnancy rate was observed (41.8%, 297/710; P < 0.01). Pregnancy rates among biopsied embryos at 3 different development stages (morula-early blastocyst, blastocyst, and expanded blastocyst) were not different. Similar results were found between embryo groups of grade 1 and 2. A significant decrease in pregnancy rate (0/10) was observed with embryos held in vitro for a longer period of time (>5 h), suggesting detrimental effects of in vitro conditions on embryo survival. The highest pregnancy rate (68.0%) was observed in recipients synchronized for the first time before being implanted with biopsied embryos. Significant decreases in such rates were found in recipients synchronized for the second or third times or those with an abortion history at the first or second synchronization-implantation treatment (P < 0.01). Better pregnancy rates (45.6%, 41/90; 46.1%, 76/165; and 45.5%, 5/11) were obtained for recipients implanted with biopsied embryos at Days 7.5, 8.0, and 8.5 post-heat detection, respectively, compared to 16% at Day 7 (3/18, P < 0.05). It is concluded that mechanical treatment (cutting) does not reduce the survival of biopsied embryos; however, cryopreservation reduces their ability to survive in vivo. The analyses also suggest that holding embryos in vitro should not be longer than 5 h unless more favorable in vitro conditions can be provided. To achieve better results of implantation of biopsied embryos, embryo transfer should be performed during 7.5–8.5 days post-estrus, and the healthy recipients synchronized for the first time should be used.

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Factors affecting pregnancy rate following embryo transfer in the cow

Theriogenology ◽

10.1016/0093-691x(87)90073-2 ◽

1987 ◽

Vol 27 (1) ◽

pp. 99-113 ◽

Cited By ~ 37

Author(s):

J.M. Sreenan ◽

M.G. Diskin

Keyword(s):

Pregnancy Rate ◽

Embryo Transfer ◽

Factors Affecting

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161 PREGNANCY RATES OBTAINED AFTER EMBRYO TRANSFER AT FIXED TIME OF IN VIVO-, IVF- AND CLONED-DERIVED EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab161 ◽

2005 ◽

Vol 17 (2) ◽

pp. 231

Author(s):

J. Lagioia ◽

M. Panarace ◽

M. Marfil ◽

M. Basualdo ◽

J. Gutierrez ◽

...

Keyword(s):

Pregnancy Rate ◽

Embryo Transfer ◽

Fixed Time ◽

Pregnancy Rates ◽

Transfer Programs ◽

Estrus Detection ◽

Group 2 ◽

Low Efficiency ◽

Group 1

The most important factor in bovine embryo transfer programs is the low efficiency in the utilization of the recipients; this low efficiency is associated with low response to synchronization protocols and failures in estrus detection. It has been shown that cows transferred at fixed time with in vivo-derived embryos resulted in high rates of recipients selected for transfer and high overall pregnancy rates (recipients pregnant/recipients treated) (Tribulo et al. 2002 Theriogenology 57, 563). An experiment was designed to evaluate the pregnancy rate in recipients transferred with in vivo (fresh and frozen), IVF, and cloned-derived embryos without estrus detection. A total of 1555 non-lactating Bos Taurus crossbred beef cows was divided into two groups. Cows from group 1 (n = 421) were synchronized with a progesterone intravaginal releasing device (1 g P4; DIB, Syntex®, Buenos Aires, Argentina) plus 2 mg of estradiol benzoate (EB) i.m. (Syntex®) on Day 0. On Day 5, they received 400 IU of eCG (Novormon 5000, Syntex®) i.m. and 150 μg of D-Cloprostenol (PGF2α) (Bioprost-D, Biotay®, Buenos Aires, Argentina). The DIB devices were removed on Day 8 and on Day 9, 1 mg of EB was injected. Day 10 was arbitrarily considered as the day of estrus. Cows from group 2 (n = 1134) received 2 doses of PGF2α 14 days apart and were checked for heat during 5 days after the second PGF2α dose. Cows of both groups were examined 7 days after estrus by ultrasonography (Pie Medical Scanner 200®) and those with a corpus luteum >10 mm of diameter were transferred nonsurgically with in vivo (fresh and frozen), IVF, and cloned-derived embryos. In group 1, 360 cows were transferred, and in group 2, 726 cows were transferred (Table 1). Pregnancy was diagnosed 23 days later by ultrasonography (Pie Medical Scanner 200®). The pregnancy rates were compared statistically between groups 1 and 2 by analysis of variance (Infostat, LSD Fisher). There was no significant statistic difference (P > 0.05) between pregnancy rate in group 1 and 2 with in vivo (fresh), IVF, and cloned-derived embryos. However, pregnancy rate of frozen in vivo-derived embryos was lower in group 1 than in group 2 (P < 0.05). Results showed that treatment using DIB combined with EB, PGF2α, and eCG associated with embryo transfer without estrus detection (group 1) had no difference in pregnancy rate when compared with the treatment where synchronization with PGF2α and heat detection were used (group 2). Another important advantage is the use the group 1 treatment for increasing the flexibility and efficiency in the management of the recipients of in vivo, IVF, and cloned-derived embryo transfer programs. Table 1. Comparison of pregnancy rates between group 1 (embryo transfer at fixed time) and group 2 (embryo transfer 7 days after estrus detection)

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