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Factors affecting frozen and fresh embryo transfer pregnancy rates in cattle
ELSEVIER
FACTORS AFFECTING FROZEN AND FRESH EMBRYO TRANSFER PREGNANCY RATES IN CATTLE John F. Hasler Fort Collins, CO 80523
ABSTRACT The effects of a large number of factors on the pregnancy rates of fresh and frozen cattle embryos were examined over a period of years at several different locations. For fresh embryos, overall pregnancy rates were 68.3% (n=9023) and 77.1% (n=2650) at different locations and time periods. Frozen-thawed embryo pregnancy rates were 56.1% (n=3616) in The Netherlands and 58.4% (n=5297) and 68.7% (n=774) for two studies in the United States. Pregnancy rates of surgical versus nonsurgical transfers were very similar. There were no differences in the pregnancy rates of beef versus dairy embryos, but the pregnancy rate was higher in daii and beef heifers and beef cows than in daii cows. Although on-farm pregnancy rates in California were higher than in the northeast United States, there was no influence of season on pregnancy rate. Estrous asynchrony between plus and minus 24 h did not affect pregnancy rate for f?ozenthawed or fresh embryos. Neither breed nor parity of recipients affected the influence of asynchrony on pregnancy rates. Embryo grade was a significant factor in pregnancy rate for both t?esh and frozen-thawed embryos, but neither embryo stage nor age was a significant factor. Pregnancy rate was not af&cted by holding embryos after flushing for up to 3 h prior to freezing. 0 2001 by Elsevier Science Inc. Key words: bovine, embryo, frozen, fresh, pregnancy
INTRODUCTION Over the past 30 years, embryo transfer (ET) in cattle has grown into an international industry in which more than 500,000 embryos annually are recovered and transferred or frozen (34). The industry started during the early 1970s and initially was based exclusively on surgical recovery and transfer of fresh embryos. Today, most recovery and transfer procedures are nonsurgical, and more than 50% of cattle embryos are frozen after recovery (34). Because ET in cattle is expensive, data on many aspects of the technology have not been generated in traditional research programs. Gn the contrary, many comprehensive studies have Acknowledgments The author thanks the entire staffs of Em Tran, Inc., Em Tran-West, Inc. and Holland Genetics, without whose help this research would not have been possible. Jennifer Williams and Sallie Vamer spent many hours helping with data entries. J.F. de La Tone-Sanchez and G.E. Seidel, Jr. were especially helpful in the statistical analysis of the data. Theriogenology 56:1401-1415,200l Q 2001 Elsevier Science Inc.
009%691XI01/$-eee front matter PII: SOOSS-691X(01)00643-4
Theriogenology
involved retrospective analysis of commercial ET operations. Data on a number of factors related to transfer of both fresh (9,10,13,24,28,30,36) and fl-ozen embryos (1,11,30,35,37) have been reported previously. However, there has been little information involving the contemporaneous transfer of t?esh and frozen embryos under similar conditions in the same program. The data in the present study were collected during the operation of two jointly managed, commercial ET companies operating in different areas of the USA. Also included are data involving embryos frozen in the USA and exported, thawed and transferred in The Netherlands. A number of factors were retrospectively analyzed in relation to pregnancy rates following transfer of fresh and frozen embryos. Among the factors analyzed were location, year, season, embryo and recipient breed, embryo age, stage and grade, recipient estrus synchrony, and several factors related to freezing embryos.
MATERIALS AND METHODS Scope of the Study This study is a retrospective analysis of data collected from ongoing ET operations at Em Tran, Inc., Elizabethtown, PA (EmT), Em Tran-West, Inc., Turlock, CA (EmT-W) and Holland Genetics (HG), Arnhem, The Netherlands. Embryos were collected nonsurgically from donor females and either frozen or transferred fresh at both EmT and EmT-W. Ail transfers involving EmT-W were in client-owned recipients on-farm in central California. At EmT, embryos were transferred either into company-owned and managed Holstein heifers in south central Pennsylvania (EmT-I) or into client-owned recipients on farms throughout the northeast United States (EmT-2). The data from HG represent results of embryos frozen at EmT and exported to HG where they were thawed and transferred. The study was divided into three different phases as follows: Study 1. During a 2-year period, from May 1986 through April 1988, embryos were collected from both dairy (85%) and beef (15%) donors under on-farm conditions. One veterinarian transferred all embryos either surgically by a paralumbar incision or nonsurgically. This study covered the transition period during which nonsurgical transfers were gradually replacing surgical transfers at EmT-W. In addition, during one year on one farm, a large number of transfers were alternated between surgical and non-surgical, utilizing an exceptionally uniform group of Holstein heifer recipients maintained in a consistent management system. Studv 2. Included were all embryos transferred by personnel at EmT and EmT-W during the years 1987, 88, 91 and 92. All transfers of fresh and frozen embryos were included in the analysis except when pregnancy results were not available. Data analyzed for each transfer included: month and year of transfer, geographical location, fresh vs frozen embryo, type of straw, breed of embryo, recipient breed and parity, embryo age, stage and quality after thawing, and estrous synchrony between donor and recipient. Study 3. Embryos were frozen at EmT fiorn 1988 to 1995 and exported to HG in the Netherlands. The following data from the time of embryo collection and freezing were analyzed: month and year of embryo collection, embryo stage and quality and the time elapsed between
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embryo collection and the start of freezing. At the time of thawing and transfer, the following additional data were recorded: month and year, recipient parity, day of recipient estrous cycle, recipient estrus synchronization method, the ownership of recipients, and the transfer team. Embryo Production Although single embryos were collected from a few donors, the majority of the donor cows and heifers in this study were superovulated with commercially available FSH. All embryos in this study resulted from the nonsurgical recovery of embryos (12) 5 to 9 days after estrus (Day 0 = es&us), and most were performed 7 or 7 l/2 d after e&us. All recoveries were collected into sterile, disposable filters (Em-Con, Immuno Systems, Spring Valley, WI). Ova and embryos were identified in the filtrate under a stereomicroscope. The study includes all tkesh and tiozen-thawed transfers at EmT and EmT-W during the calendar years 1987, 88, 91 and 92. Data on frozen-thawed transfers included only embryos cozen by either EmT or EmT-W. Data from HG include only embryos tiozen by EmT during the years 1989-95 and exported to The Netherlands. Embryos Embryos were evaluated at x 50 under a stereomicroscope and placed in PBS containing 10% heat-inactivated new-born calf serum and maintained at ambient temperature. Embryos designated for fresh transfer were held for 1 to 10 h prior to transfer. Embryos to be frozen were held for 1 to 4 h prior to freezing. Embryos were evaluated for stage of embryonic development, as follows: morula (M) = embryo that had undergone compaction; early blastocyst (EB) = embryo with blastocoel comprising less than 50% of the embryonic mass; mid-blastocyst (MB) = embryo with blastocoel comprising more than 50% of the embryonic mass and no thinning of the zona pellucida; expanded blastocyst (XP) = embryo with increased overall diameter and a distinct thinning of the zona pellucida; hatched blastocyst (HP) = blastocyst-stage embryo freed of the zona pellucida. Embryos were evaluated for grade as follows: 1 = embryos at the expected stage of development relative to age, with no detectable defects; 2 = embryos at the expected stage of development with only trivial defects, such as a few small extruded cells, a slightly irregular shape or a few vesicles; 3 = embryos with obvious defects, such as being slightly behind in development, numerous extruded cells, but with more than 50% of the cellular mass intact; 4 = embryos with less than 50% of the cellular mass intact, oflen severely retarded in development. Embryo Freezing and Thawing A few embryos thawed during this study were t?ozen in lmL-glass ampules prior to 1985. Subsequently, all embryos were frozen in OS-mL plastic straws until January 1991 and in 0.25-d straws after that date. Embryos were equilibrated for 10 to 20 min at ambient temperature in a cryoprotectant solution composed of Dulbecco’s PBS (Gibco BRL, Grand Island, NY) containing 4 g/L BSA and 10% v/v glycerol. Following equilibration, straws were
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placed in a programmable i?eezer maintained at -4.5 to -5°C and seeded within one min. The straws were held at seeding temperature for 15 min and then cooled at -05°C per min, before being plunged into liquid nitrogen at -32°C. Embryos were thawed in a 35°C water bath, removed from straws and moved through the following solutions at 5 min intervals: 1) PBS containing 6.7% glycerol + 0.3M sucrose, 2) 3.3% glycerol + 0.3M sucrose and 3) 0.3M sucrose. Then embryos were rinsed three times in PBS and loaded in 0.25~rnL straws for transfer. A maximum of 6 embryos was thawed at any one time prior to transfer. Recipients Recipients owned by Em Tran, Inc. were exclusively virgin Holstein heifers purchased at 14 to 18 months of age, weighing 340 to 410 kg. These heifers were observed twice daily for onset of either naturally occurring or prostaglandin-induced estrus. Clients of both Em Tran, Inc. and Em Tran-West Inc. provided cows and heifers of various beef and dairy breeds as recipients. Clients had the obligation of observing and recording of estrus. Transfers at Holland Genetics were primarily into virgin Holstein heifers that had been observed in natural estrus. Transfers In all but rare cases, recipients only were used if they had been observed in standing estrus and possessed a palpable corpus luteum (CL) at the time of transfer. Single embryos were transferred tr anscervically with a standard l/4 mL embryo transfer instrument into the uterine horn ipsilateral to the CL. Depending on variables, such as the availability of recipients, the number of embryos available to be transferred and specific economic factors, embryos were transferred into recipients that had been in estrus ranging from 48 h before the donor (plus asynchrony) to 48 h after the donor. Approximately 50 to 60 d following transfer, recipients were examined for pregnancy by rectal palpation. Statistical Analysis Studv 1. Data were analyzed by chi-square with the Fisher-Yates correction. Studv 2. From the original data set of 14,699 animals, 374 recipients with no pregnancy information were excluded from the analysis, along with 5 recipients that died between ET and pregnancy diagnosis. Data for fresh and tiozen embryos were evaluated in separate analyses. Because subclass pregnancy rate means were nearly all in the 40 to 70% range, transformations were not needed to normalize data. To decrease complexity of statistical analyses, the ANOVAs were done in stages because of the large number of factors and interactions. The first stage of analysis considered all main effects. The second stage included all factors with significant main effects from the first stage plus all first order interactions. The final ANOVA that was used included the same main effects as the second stage analysis plus all significant (p
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as a polynomial regression equation. Arithmetic means are presented in tables instead of least squares means because they are more illustrative of actual pregnancy rates; least squares means were much lower than actual means as would be expected. Difkences among means were tested for significance by Tukey’s test. In a few cases clearly indicated in table footnotes, chisquare analysis was used as an adjunct statistical procedure. Studv 3. Data were analyzed by factorial, least squares ANOVA.
RESULTS Study 1 The overall pregnancy rate during the two-year period at EmT-W was 77.1% for 2650 lksh embryo transfers and 68.7% for 774 frozen-thawed embryo transfers. When heifers were used as recipients, pregnancy rates between surgical and nonsurgical transfers were not different whether the embryos were fresh or fiozen (Table 1). Likewise, the pregnancy rate in cows was not different between surgical and non-surgical transfers of fresh embryos. Cows exhibited a lower pregnancy rate, however, when fiozcn embryos were transferred non-surgically compared to surgically. The pregnancy rate for cows was lower than for heifers for fresh embryos transferred either surgical or non-surgical, but not for tkozen embryos.
Table 1. Pregnancy rates following surgical or nonsurgical transfer of fresh or cozen-thawed embryos to cows or heifers. Fresh Frozen No. Transfers No. Transfers % Pregnant % Pregnant Surgical Heifers 1485 586 70.8’ 79.9a cows 90 491 69.7b 71.1’ Nonsurgical Heifers 590 78.8“ 72 59.7 cows 84 60.7b 26 38.5d 4b*c*dValuesin columns without common superscripts differ significantly (a vs b: P
Of the transfers in Table 1, 580 were performed on one fkm during a period of approximately one year. The mean quality grade of the 213 embryos transferred surgically was 1.61, compared to 1.62 for 367 embryos that were transferred nonsurgically. There was no significant difference between the 85.0% surgical and 79.3% non-surgical pregnancy rates on this farm
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Study 2 The overall pregnancy rate resulting from transfer of 9023 fresh embryos was 68.3%, compared to 58.4% (P
Table 2. The effect of geographic location, year and season on pregnancy rates of fresh and frozen-thawed embryos Fresh Frozen No. Transfers % Pregnant No. Transfers % Pregnant Year 1987 2512 68.6 1174 61.3 1988 2716 67.3 1290 58.5 1991 1960 70.1 1479 57.5 1992 1835 67.2 1354 56.7 Location EmT-W 5345 73.2a 2879 61.5’ EmT- 1 1173 62.2b 707 56.0 EmT-2 2505 61.0b 1711 54.1b Season Spring 2718 68.8 1394 58.2 Summer 2004 67.7 1691 60.9 Fall 1907 67.8 883 58.9 Winter 2394 68.5 55.1 1329 “,bValues in columns without common superscripts differ significantly (PcO.05)
There was no difference in pregnancy rates between embryos of dairy and beef origin whether they were transferred fresh or Cozen-thawed (Table 3). The pregnancy rate of dairy cowrecipients was lower (P
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effect of synchrony on the pregnancy rate of t?esh but not frozen-thawed embryos. embryos, the linear and quadratic effects significantly differed from zero (PcO.05).
For fresh
Table 3. The effect of embryo breed and recipient breed and parity on pregnancy rates of fresh and frozen-thawed embryos Fresh Frozen % Pregnant No. Transfers % Pregnant No. Transfers Donors Dairy 7457 68.5 4038 58.8 Beef 1566 67.3 1259 57.2 Recipients Dairy Heifers 6612 70.58 3477 60.9” Dairy Cows 844 52.gb 518 47.1b Beef Heifers 267 65.9’ 252 60.0’ Beef Cows 835 68.68 461 58.6” “Sbvalues in columns without common superscripts differ significantly (P
-e
Fresh
90 = 80 c” 70 p 80 a 50 $ 40 30 48
38
24 Plus
12
0
Synchrony
-12 (hours)
-24
-38
48
Minus
Figure 1. Effect of recipient-donor estrus synchrony on pregnancy rate following transfer of fresh and frozen-thawed embryos. Plus asynchrony=recipient in estrus before donor; Minus asynchrony=recipient in estrus after donor. Left to right, n for fksh embryos: 54, 149,979, 1836,3524, 1377,922, 142 and 13; for frozen embryos: 11, 125, 707, 1298,2124,753,227 and 26. The relationship between estrus synchrony and pregnancy rate following transfer of both fresh and frozen embryos into dairy heifers and cows and beef cows is shown in Figure 2. Although the pregnancy rate was lower in dairy cows than in beef cows or dairy heifers, the curves were very similar.
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I
q-Dairy
-
heifers
:r
48
Plus
36
24
+Beefcows
12
0
*Dairy
-12
Synchrony (hours)
-24
cows
-36
I
-48
Minus
Figure 2. Effect of recipient-donor estrus synchrony on pregnancy rate following transfer of fresh and frozen-thawed embryos (combined totals) into dairy heifers and cows and beef cows. Plus asynchrony = recipient in estrus before donor; h4inus asynchrony= recipient in estrus after donor. Left to right, n for dairy heifers: 40, 182, 1224,2240, 4026, 1434, 804, and 108; for dii cows: 137, 252, 548, 233 and 127; for beef cows: 129,298,490,236 and 103.
There were no differences in pregnancy rates of embryos frozen in 0.5 mL versus 0.25 mL straws (Table 4). Pregnancy rates were lower, however, following transfer of embryos that were frozen in glass ampules, than embryos frozen in either 0.5 or 0.25 mL straws.
Table 4. The effect of straw size and glass ampules on the pregnancy rate of recipients. No. Transfers % Pregnant Embryo container 0.5 mL straws 3245 58.9” 0.25 mL straws 2056 57.5a Glass ampules 62 30.6b “%alues in columns without common superscripts differ significantly (P
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Table 5. Effects of tksh and frozen-thawed embryo grade, stage of development and age on pregnancy of recipients Frozen Fresh % Pregnant No. Transfers % Pregnant No. Transfers Embrvo Grade 1 2 3
4163 3156 1641
73.2’ 68.3b 56.3’
2482 2329 454
61
47.5c
22
62.8’ 56.8b 43.6’
4 Embrvo Stage M EB MB xl3 HB
36.4b,”
5633 1978 995 391 25
66.9 70.3 70.9 71.4 56.0
3576 1140 478 93 __^
57.7 61.2 57.9 50.5 ---
Embrvo Ane (dl 5 5.5 6 6.5 7 7.5 8 8.5 9
13 13 225 490 6810 1051 339 36 23
76.9 61.5 65.8 70.0 68.8 65.4 67.6 72.2 34.8
_-_ --18 84 3794 1224 129 -___-
___ --_ 55.6 61.9 58.3 60.0 50.4 --___
a,b*cgdValues in columns without common superscripts differ significantly (P~0.05)
Study 3 Between 1988 to 1995, a total of 3616 embryos frozen at EmT were thawed and transferred by personnel at HG, resulting in 2030 pregnancies (56.1%). The location of transfers was divided between recipients owned and housed by HG (n-3 122) and recipients on private farms in The Netherlands (t&94). Analysis of variance showed that the following factors did not significantly affect pregnancy rate: year or season of Ikeezing, year or season of transfer, location of transfer, parity of recipient and transfer team. Likewise, the time between flushing and freezing did not affect pregnancy rate (Table 6). In addition, interactions were not significant (P
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Table 6. Effects of day of recipient estrous cycle, embryo grade and stage, and the time from flushing to lkeezing on pregnancy rate of frozen-thawed embryos No. Transfers % Pregnant Day of Recipient Estrous Cycle 53.6b 8 (plus)’ 774 59.0” 7 (0) 2015 48.9b 6 (minus) 468 Embryo Grade2 57.8’ 1 1723 54.3c 2 788 37.9* 3 253 Embryo Stage 54.3f M 1997 59.7e EB 494 52.1’ MB 257 Time Between Flushing and Freezing (min) 54.1 0 -30 85 56.0 30 -60 418 56.0 61-90 722 56.2 91-120 507 53.0 121-150 264 55.7 151-180 106 ‘Estrous synchrony, 2Before freezing, ‘*b~c~4e%aluesin columns without common superscripts differ significantly (a vs b: P
DISCUSSION During the early years of the ET industry, it was reported that pregnancy rates from NS transfer were lower than those from surgical transfer (20,28,31). One subsequent report also showed a higher pregnancy rate resulting tiom surgical transfer (33), while others reported no difference (2,lO). The present study indicated that comparable pregnancy rates can be achieved in heifers with both methods of transfer. Nevertheless, the data suggest that pregnancy rates may favor surgical transfers in cows. The 8.4 and 9.9 percentage point differences between the pregnancy rates of hesh and tiozen embryos in Studies 1 and 2 are similar to the range of differences for tiesh and t?ozen embryos (8.5 to 14) previously reported (8,15,19,30,32). In Study 2, there were similar differences in pregnancy rates between t+esh and frozen embryos at each grade level. This suggests that the decrease in pregnancy rate associated with frozen-thawed embryos is due to damage that is not visually obvious based on quality grading procedures.
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Data from Study 1 indicate that higher pregnancy rates were achieved in heifers than cows. Approximately 85% of the animals in this study were Holsteins, while the balance were a mixture of beef breeds. Similarly, in Study 2, transfer of either fresh or frozen embryos resulted in higher pregnancy rates in dairy heifers and in both beef heifers and cows than in dairy cows. Other investigators have also reported a higher pregnancy rate in dairy heifers than in dairy cows following NS transfer of either fresh (8) or frozen embryos (8,ll) whereas still others either reported no difference (7), or equivocal results (5) that were attributed to nutritional deficiencies in the cows. The differences between daii heifers and cows in the present study may be due to the higher stress levels associated with lactation that are imposed upon cows in many dairy facilities. It has been well documented that reproductive efficiency in dairy cattle has declined over the last several decades (6,27). Conception rate declined in dairy cows in the USA from 66% in 1951 to 50% in 1973, while average milk production increased by 33% (6). It is noteworthy that during this period, conception rate in virgin dairy heifers did not decline during this period and, if fact, heifers and cows were equally fertile to AI in 1954 (6). This suggests that the decline in cows is related to the stress of greater milk production and is not due to genetic selection for lower fertility. Nevertheless, the decline in fertility appears to be associated with a rise in genetic selection for milk production (18,23). Pryce et al. (23) reported that body condition score recorded 1 month after calving had the largest genetic correlation with calving interval. Modifications in diet may provide the greatest aid in counteracting the decrease in fertility that is related to genetic selection for increased milk production (3,23). Obviously, nutritional and lactational stress can also be of considerable importance in postpartum beef cows. However, neither the present study nor the study by Wright (36) showed any difference in pregnancy rates between beef heifers and cows, The lack of yearly differences in pregnancy rates of fresh or frozen embryos in the present study is consistent with some previous reports (9,ll). However, Hasler et al. (13) reported some yearly differences in pregnancy rate following surgical transfer into Holstein heifers over a 7-year period. In the present study, the interaction between year and location has no obvious biological explanation. It is somewhat surprising that there was no influence of season within location on pregnancy rate, considering the contrast in climate represented by central California and the northeast United States. However, other studies that included embryo transfers in Japan and in both the central and southwest United States, also failed to show any seasonal infhtences on pregnancy rate (9,11,24). Furthermore, Putney et al. (24) did not detect an influence of season on pregnancy rates following embryo transfas in the Southwest USA even though monthly mean environmental temperatures ranged Tom 7.8 to 28.6”C. The study by Lindner and Wright (16) is frequently used as a reference for embryo quality relative to pregnancy rate. Subsequently, the IETS published a grading system for bovine embryos (26) in which the separate excellent and good categories of Lindner and Wright (16) were combined into a single grade designated as number 1. Similarly, IETS Grade 2 embryos represent Lindner and Wright’s third category of fair embryos. The rating system in the present study more closely resembles that used by Lindner and Wright (16). Consistent criteria have not been employed for grading the morphological quality of cattle embryos in the literature (9,10,13,16,26,30,36). Nevertheless, the majority of these studies indicated significant differences in pregnancy rate among different grades of either frozen and/or &esh embryos.
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However, most studies have combined excellent and good embryos into one grade and, as a consequence not been able to relate the two grades to pregnancy rates. The present study is both supported (10,13) and contradicted (16,21,30) by several other studies in demonstrating differences in pregnancy rates between excellent and good quality embryos. Although the IETS combines the two grades into one for the purposes of international standardization, there may be situations where there are economic advantages to handle the two grades separately. Assessment of cattle embryo developmental stages is not subject to as much variation as is quality grading. Many studies have not involved sufficiently large sample sizes to detect small differences among stages. Nevertheless, in contrast to the present study, a number of studies have shown the transfer of t?esh blastocysts generally resulted in higher pregnancy rates than morulae (10,13,24,28,36). Other published reports have shown a similar pregnancy rate for morulae and blastocysts (21), a lower pregnancy rate for mid- and expanded-blastocysts than morulae (1 l), or no differences (35). Synchrony between the age of the embryo and the estrous cycle of the recipient has long received attention as potentially affecting the success rate of ET. The present study indicated differences (PcO.05) in pregnancy rates relative to estrous synchrony based on ANOVA. It is very clear, however, that for practical application in the field, 24 h plus or minus asynchrony resulted in pregnancy rates comparable to zero synchrony with either fresh or frozen-thawed embryos. There is no indication that frozen-thawed embryos are any more or less sensitive to asynchrony than are fresh embryos. Some previous studies on synchrony with fresh embryos have indicated that beef recipients were apparently more sensitive to asynchrony (10,24,28,35) than dairy heifers (9,14). There were no differences in pregnancy rate relative to asynchrony in the present study among beef cows, dairy cows and daii heifers. With rare exceptions (17), the initial successes with commercial freezing of cattle embryos involved the use of glass ampules (22,29). As pointed out in a recent review (25), however, higher rates of zona fkacture were associated with the use of glass ampules compared to plastic straws. Plastic straws have largely replaced glass ampules as the container of choice for freezing cattle embryos. The results of the present study show that there is no advantage of the size of the straw on embryo survival, but both sizes of straws resulted in higher pregnancy rates than glass ampules. The time between embryo collection and keezing is an important practical consideration in commercial ET. Previous studies (22,36) have shown that there was a drop in pregnancy rate when embryos were held for more than 4 h prior to freezing. Otter (21) reported that pregnancy rate decreased for embryos held for between 2 to 4 or 4 to 6 h, compared to 0 to 2 h prior to lkeezing. The present study showed no effect of time to hezing at intervals of 30 min up to 3 h following embryo collection. Virtually all the &zing, however, was conducted in a laboratory with an ambient temperature that was maintained below 28OC in summer. It is possible that maintainmg embryos at higher temperatures, such as those encountered in on-farm situations in summer would result in lower embryo survival rates. The cryoprotectant used for fkezing embryos in the present study was 10% glycerol, whereas 1.5 M ethylene glycol has become the predominant cryoprotectant in the commercial ET
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industry in the last few years. Published reports have indicated that survival is similar for both in vitro- (14) and in vivo-derived embryos (1,8,11,15) frozen in either of the two cryoprotectants. Consequently, it is reasonable to assume that the influence on pregnancy rate of the factors examined in this study will also apply to embryos frozen in ethylene glycol.
CONCLUSIONS There was no evident loss of pregnancy rate with the transition Iiom surgical to nonsurgical transfer of embryos in a commercial ET program Location and year, but not season affected pregnancy rate. There was a loss in pregnancy rate of approximately 10 to 13 percentage points between fresh and frozen-thawed embryos of comparable grades. Dairy cows sustained a lower pregnancy rate than daii heifers or beef heifers or cows. Although embryo quality was of paramount importance in determining pregnancy rate for both fresh and frozen-thawed embryos, embryo stage of development did not influence pregnancy rate. Estrous asynchrony affected fresh and frozen-thawed embryos pregnancy rates in a similar fashion, with no evident affect when synchrony was within 24 h plus or minus of zero.
REFERENCES 1.
Arreseigor, CJ, Sisul, A, Arreseigor, AE, Stahringer, RC. Effect of cryoprotectant, thawing method, embryo grade and breed on pregnancy rates of cryopreserved bovine embryos.. Theriogenology 1998;49: 160 abstr. 2. Baker, AA, Kobayaski, G, Jillella, DA. Comparison of the pregnancy rate following nonsurgical and surgical transfer and visual grading of bovine embryos on farms in southeastern Queensland. Theriogenology 1983;19:111 abstr. 3. Boland, MP, Lonergan, P, O’Callaghan. Effect of nutrition on endocrine parameters, ovarian physiology, and oocytes and embryo development. Theriogenology 2001;55:1323-1340. 4. Breuel, KF, Baker, RD, Butcher, RL, Townsend, EC, Inskeep, EK, Dailey, RA, Lemer, SP. Effects of breed, age of donor and dosage of follicle stimulating hormone on the superovulatory response of beef cows. Theriogenology 1991;36:241-255. Broadbent, PJ, Stewart, M, Dohnan, DF. Recipient management and embryo transfer. Theriogenology 1991;35:125-139. Butler, WR, Smith, RD. Interrelationships between energy balance and postpartum reproductive function in dairy cattle. J. Dairy Sci. 1989;72:767-783. Callesen, H, Bak, A, Greve, T. Embryo recipients: dairy cows or heifers? Proc. of the 10’ Scientific Meeting of the AETE, Lyon, France, 1994;125-135. Chagas, ESJ, Cidadao, MR, Lopes, DCL. Effect of parity and type of estrus of recipient on pregnancy rate following embryo transfer in dairy cattle. Proc. of the 15* Scientific Meeting of the AETE, Lyon, France, 1999;132 abstr. 9. Coleman DA, Dailey, RA, Leffel, RE, Baker, RD. Estrous synchronization and establishment of pregnancy in bovine embryo transfer recipients. J Dairy Sci. 1987;70:858866. 10. Donaldson, LE. Matching of embryo stages and grades with recipient oetrous synchrony in bovine embryo transfer. Vet Ret 1985;489-491.
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11. Do&i, 0, Yamamoto, Y, Saga, H, Yoshiba, N, Kano, N, Maeda, J, Miyata, K, Yamauchi, A, Tominaga, K, Oda, Y, Nakashima, T, Inohae, S. Direct transfer of bovine embryos fiozenthawed in the presence of propylene glycol or ethylene glycol under on-farm conditions in an integrated embryo transfer program. Theriogenology 1998;49: 105 1- 1058. 12. Elsden, RP, Hasler, JF, Seidel, GE, Jr. Nonsurgical recovery of bovine eggs. Theriogenology 1976;6:523-532. 13. Hasler, JF, McCauley, AD, Lathrop, WF, Foote, RH. Effect of donor-embryo-recipient interactions on pregnancy rate in a large-scale bovine embryo transfer program. Theriogenology 1987;139-168. 14. Hasler, JF, Hurtgen, PJ, Jin, ZQ, Stokes, JE. Survival of IVF-derived bovine embryos frozen in glycerol or ethylene glycol. Theriogenology 1997;48:563-579. 15. Lester, TD, McNew, RW, Rorie, RW. Use of electronic estrous detection to evaluate the effect of embryo-recipient synchrony on pregnancy rate in cattle. Theriogenology 1999;5 1:265 abstr. 16. Lindner, GM, Wright, RW, Jr. Bovine embryo morphology and evaluation. Theriogenology 1983;20:407-416. 17. Massip, A, Van der Zwahnen, P, Ectors, F, De Coster, R, D’leteren, C, Hanzen, C. Deep freezing of cattle embryos in glass ampules or French straws. Theriogenology 1979;12:7984. 18. Nebel, RL, McGilliard, ML. Interactions of high milk yield and reproductive performance in dairy cows. J. Dairy Sci. 1993;76:3257-3268. 19. Nelson, CF, Nelson, LD. Cryopreservation of 7- to 9-day bovine embryos. Theriogenology 1988;29:28 1 abstr. 20. Newcomb, R, Christie, WB, Rowson, LEA. Comparison of the fetal survival rate in heifers after the transfer of an embryo surgically to one uterine horn and non-surgically to the other. J. Reprod. Fert. 1978;52:395-397. 21. Otter, T. Pregnancy rate of fresh and frozen-thawed cattle embryos. Proc. of the lo* Scientific Meeting of the AETE, Lyon, France, 1994;228 abstr. 22. Pettit, WH, Jr. Commercial freezing of bovine embryos in glass ampules. Theriogenology 1985;23:13-16. 23. Pryce, JE, Coffey, MP, Brotherstone, S. The genetic relationship between calving interval, body condition score and linear type and management traits in registered Holsteins. J. Dairy Sci. 2000;83:2664-2671. 24. Putney, DJ, Thatcher, WW, Drost, M, Wright, JM, DeLorenzo, MA. Influence of environmental temperature on reproductive performance of bovine embryo donors and recipients in the southwest region of the United States. Theriogenology 1988;30:905-922. 25. Rall, WF. Cryopreservation of mammalian embryos, gametes and ovarian tissues. In: Wolf, DP and M Zelinski-Wooten (eds), Assisted Fertilization and Nuclear transfer in Mammals. Totowa, New Jersey: Humana Press, 200 1; 173- 187. 26. Robertson, I, Nelson RE. Certification and identification of the embryo. In: Stringfellow DA, Seidel, SM (eds), Manual of the International Embryo Transfer Society. Savoy, IL:IETS, 1998;103-134. 27. Rogers, GW, Banos, G, Sander-Nielsen, U. Genetic correlations among protein yield productive life, type traits from the United States and diseases other than mastitis Tom Denmark and Sweden. J. Dairy Sci. 1999;82:1331-1338.
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28. Schneider, HJ, Castleberry, RS, Griffi 29.
30. 31. 32. 33. 34.
35.
36. 37.
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JL. Commercial aspects of bovine embryo transfer. Theriogenology 1980;13:73-85. Shea, BF, Janzen, RE, McAlister, RJ, McDermand, DP. Freezing of bovine embryos: effects of embryo quality, time from thawing to transfer and number frozen per vial. Theriogenology 1983;20:205-212. Spell, AR, Beal, WE, Corah, LR, Lamb, GC. Evaluating recipient and embryo factors that affect pregnancy rates of embryo transfer in beef cattle. Theriogenology 2001;56:287-297. Sreenan, JM. Non-surgical embryo transfer in the cow. Theriogenology 1978;9:69-83. Sreenan, JM, Dislcm, MG. Factors affecting pregnancy rate following embryo transfer in the cow. Theriogenology 1987;27:99-113. Takeda, T, Hallowell, SV, McCauley, AD, Hasler, JF. Pregnancy rates with intact and split bovine embryos transferred surgically and nonsurgically. Theriogenology 1986;25:204. Thibier, M. The IETS statistics of embryo transfers in livestock in the world for the year 1999: A new record for bovine in vivo-derived embryos transferred. In: Wheeler, M (ed), Embryo Transfer Newsletter. Savoy, IL: 2000;18:24-28. van Wagtendonk-de Leeuw, AM, den Daas, JHG, Rail, WF. Field trial to compare pregnancy rates of bovine embryo cryopreservation methods: vitrification and one-step dilution versus slow tieezing and three-step dilution. Theriogenology 1997;48:1071-1084. Wright, JM. Non-Surgical embryo transfer in cattle: embryo-recipient interactions. Theriogenology 1981;15:43-56. Wright, JM. Commercial freezing of bovine embryos in straws. Theriogenology 1985;23:1729.
FACTORS AFFECTING FROZEN AND FRESH EMBRYO TRANSFER PREGNANCY RATES IN CATTLE John F. Hasler Fort Collins, CO 80523
ABSTRACT The effects of a large number of factors on the pregnancy rates of fresh and frozen cattle embryos were examined over a period of years at several different locations. For fresh embryos, overall pregnancy rates were 68.3% (n=9023) and 77.1% (n=2650) at different locations and time periods. Frozen-thawed embryo pregnancy rates were 56.1% (n=3616) in The Netherlands and 58.4% (n=5297) and 68.7% (n=774) for two studies in the United States. Pregnancy rates of surgical versus nonsurgical transfers were very similar. There were no differences in the pregnancy rates of beef versus dairy embryos, but the pregnancy rate was higher in daii and beef heifers and beef cows than in daii cows. Although on-farm pregnancy rates in California were higher than in the northeast United States, there was no influence of season on pregnancy rate. Estrous asynchrony between plus and minus 24 h did not affect pregnancy rate for f?ozenthawed or fresh embryos. Neither breed nor parity of recipients affected the influence of asynchrony on pregnancy rates. Embryo grade was a significant factor in pregnancy rate for both t?esh and frozen-thawed embryos, but neither embryo stage nor age was a significant factor. Pregnancy rate was not af&cted by holding embryos after flushing for up to 3 h prior to freezing. 0 2001 by Elsevier Science Inc. Key words: bovine, embryo, frozen, fresh, pregnancy
INTRODUCTION Over the past 30 years, embryo transfer (ET) in cattle has grown into an international industry in which more than 500,000 embryos annually are recovered and transferred or frozen (34). The industry started during the early 1970s and initially was based exclusively on surgical recovery and transfer of fresh embryos. Today, most recovery and transfer procedures are nonsurgical, and more than 50% of cattle embryos are frozen after recovery (34). Because ET in cattle is expensive, data on many aspects of the technology have not been generated in traditional research programs. Gn the contrary, many comprehensive studies have Acknowledgments The author thanks the entire staffs of Em Tran, Inc., Em Tran-West, Inc. and Holland Genetics, without whose help this research would not have been possible. Jennifer Williams and Sallie Vamer spent many hours helping with data entries. J.F. de La Tone-Sanchez and G.E. Seidel, Jr. were especially helpful in the statistical analysis of the data. Theriogenology 56:1401-1415,200l Q 2001 Elsevier Science Inc.
009%691XI01/$-eee front matter PII: SOOSS-691X(01)00643-4
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involved retrospective analysis of commercial ET operations. Data on a number of factors related to transfer of both fresh (9,10,13,24,28,30,36) and fl-ozen embryos (1,11,30,35,37) have been reported previously. However, there has been little information involving the contemporaneous transfer of t?esh and frozen embryos under similar conditions in the same program. The data in the present study were collected during the operation of two jointly managed, commercial ET companies operating in different areas of the USA. Also included are data involving embryos frozen in the USA and exported, thawed and transferred in The Netherlands. A number of factors were retrospectively analyzed in relation to pregnancy rates following transfer of fresh and frozen embryos. Among the factors analyzed were location, year, season, embryo and recipient breed, embryo age, stage and grade, recipient estrus synchrony, and several factors related to freezing embryos.
MATERIALS AND METHODS Scope of the Study This study is a retrospective analysis of data collected from ongoing ET operations at Em Tran, Inc., Elizabethtown, PA (EmT), Em Tran-West, Inc., Turlock, CA (EmT-W) and Holland Genetics (HG), Arnhem, The Netherlands. Embryos were collected nonsurgically from donor females and either frozen or transferred fresh at both EmT and EmT-W. Ail transfers involving EmT-W were in client-owned recipients on-farm in central California. At EmT, embryos were transferred either into company-owned and managed Holstein heifers in south central Pennsylvania (EmT-I) or into client-owned recipients on farms throughout the northeast United States (EmT-2). The data from HG represent results of embryos frozen at EmT and exported to HG where they were thawed and transferred. The study was divided into three different phases as follows: Study 1. During a 2-year period, from May 1986 through April 1988, embryos were collected from both dairy (85%) and beef (15%) donors under on-farm conditions. One veterinarian transferred all embryos either surgically by a paralumbar incision or nonsurgically. This study covered the transition period during which nonsurgical transfers were gradually replacing surgical transfers at EmT-W. In addition, during one year on one farm, a large number of transfers were alternated between surgical and non-surgical, utilizing an exceptionally uniform group of Holstein heifer recipients maintained in a consistent management system. Studv 2. Included were all embryos transferred by personnel at EmT and EmT-W during the years 1987, 88, 91 and 92. All transfers of fresh and frozen embryos were included in the analysis except when pregnancy results were not available. Data analyzed for each transfer included: month and year of transfer, geographical location, fresh vs frozen embryo, type of straw, breed of embryo, recipient breed and parity, embryo age, stage and quality after thawing, and estrous synchrony between donor and recipient. Study 3. Embryos were frozen at EmT fiorn 1988 to 1995 and exported to HG in the Netherlands. The following data from the time of embryo collection and freezing were analyzed: month and year of embryo collection, embryo stage and quality and the time elapsed between
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embryo collection and the start of freezing. At the time of thawing and transfer, the following additional data were recorded: month and year, recipient parity, day of recipient estrous cycle, recipient estrus synchronization method, the ownership of recipients, and the transfer team. Embryo Production Although single embryos were collected from a few donors, the majority of the donor cows and heifers in this study were superovulated with commercially available FSH. All embryos in this study resulted from the nonsurgical recovery of embryos (12) 5 to 9 days after estrus (Day 0 = es&us), and most were performed 7 or 7 l/2 d after e&us. All recoveries were collected into sterile, disposable filters (Em-Con, Immuno Systems, Spring Valley, WI). Ova and embryos were identified in the filtrate under a stereomicroscope. The study includes all tkesh and tiozen-thawed transfers at EmT and EmT-W during the calendar years 1987, 88, 91 and 92. Data on frozen-thawed transfers included only embryos cozen by either EmT or EmT-W. Data from HG include only embryos tiozen by EmT during the years 1989-95 and exported to The Netherlands. Embryos Embryos were evaluated at x 50 under a stereomicroscope and placed in PBS containing 10% heat-inactivated new-born calf serum and maintained at ambient temperature. Embryos designated for fresh transfer were held for 1 to 10 h prior to transfer. Embryos to be frozen were held for 1 to 4 h prior to freezing. Embryos were evaluated for stage of embryonic development, as follows: morula (M) = embryo that had undergone compaction; early blastocyst (EB) = embryo with blastocoel comprising less than 50% of the embryonic mass; mid-blastocyst (MB) = embryo with blastocoel comprising more than 50% of the embryonic mass and no thinning of the zona pellucida; expanded blastocyst (XP) = embryo with increased overall diameter and a distinct thinning of the zona pellucida; hatched blastocyst (HP) = blastocyst-stage embryo freed of the zona pellucida. Embryos were evaluated for grade as follows: 1 = embryos at the expected stage of development relative to age, with no detectable defects; 2 = embryos at the expected stage of development with only trivial defects, such as a few small extruded cells, a slightly irregular shape or a few vesicles; 3 = embryos with obvious defects, such as being slightly behind in development, numerous extruded cells, but with more than 50% of the cellular mass intact; 4 = embryos with less than 50% of the cellular mass intact, oflen severely retarded in development. Embryo Freezing and Thawing A few embryos thawed during this study were t?ozen in lmL-glass ampules prior to 1985. Subsequently, all embryos were frozen in OS-mL plastic straws until January 1991 and in 0.25-d straws after that date. Embryos were equilibrated for 10 to 20 min at ambient temperature in a cryoprotectant solution composed of Dulbecco’s PBS (Gibco BRL, Grand Island, NY) containing 4 g/L BSA and 10% v/v glycerol. Following equilibration, straws were
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placed in a programmable i?eezer maintained at -4.5 to -5°C and seeded within one min. The straws were held at seeding temperature for 15 min and then cooled at -05°C per min, before being plunged into liquid nitrogen at -32°C. Embryos were thawed in a 35°C water bath, removed from straws and moved through the following solutions at 5 min intervals: 1) PBS containing 6.7% glycerol + 0.3M sucrose, 2) 3.3% glycerol + 0.3M sucrose and 3) 0.3M sucrose. Then embryos were rinsed three times in PBS and loaded in 0.25~rnL straws for transfer. A maximum of 6 embryos was thawed at any one time prior to transfer. Recipients Recipients owned by Em Tran, Inc. were exclusively virgin Holstein heifers purchased at 14 to 18 months of age, weighing 340 to 410 kg. These heifers were observed twice daily for onset of either naturally occurring or prostaglandin-induced estrus. Clients of both Em Tran, Inc. and Em Tran-West Inc. provided cows and heifers of various beef and dairy breeds as recipients. Clients had the obligation of observing and recording of estrus. Transfers at Holland Genetics were primarily into virgin Holstein heifers that had been observed in natural estrus. Transfers In all but rare cases, recipients only were used if they had been observed in standing estrus and possessed a palpable corpus luteum (CL) at the time of transfer. Single embryos were transferred tr anscervically with a standard l/4 mL embryo transfer instrument into the uterine horn ipsilateral to the CL. Depending on variables, such as the availability of recipients, the number of embryos available to be transferred and specific economic factors, embryos were transferred into recipients that had been in estrus ranging from 48 h before the donor (plus asynchrony) to 48 h after the donor. Approximately 50 to 60 d following transfer, recipients were examined for pregnancy by rectal palpation. Statistical Analysis Studv 1. Data were analyzed by chi-square with the Fisher-Yates correction. Studv 2. From the original data set of 14,699 animals, 374 recipients with no pregnancy information were excluded from the analysis, along with 5 recipients that died between ET and pregnancy diagnosis. Data for fresh and tiozen embryos were evaluated in separate analyses. Because subclass pregnancy rate means were nearly all in the 40 to 70% range, transformations were not needed to normalize data. To decrease complexity of statistical analyses, the ANOVAs were done in stages because of the large number of factors and interactions. The first stage of analysis considered all main effects. The second stage included all factors with significant main effects from the first stage plus all first order interactions. The final ANOVA that was used included the same main effects as the second stage analysis plus all significant (p
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as a polynomial regression equation. Arithmetic means are presented in tables instead of least squares means because they are more illustrative of actual pregnancy rates; least squares means were much lower than actual means as would be expected. Difkences among means were tested for significance by Tukey’s test. In a few cases clearly indicated in table footnotes, chisquare analysis was used as an adjunct statistical procedure. Studv 3. Data were analyzed by factorial, least squares ANOVA.
RESULTS Study 1 The overall pregnancy rate during the two-year period at EmT-W was 77.1% for 2650 lksh embryo transfers and 68.7% for 774 frozen-thawed embryo transfers. When heifers were used as recipients, pregnancy rates between surgical and nonsurgical transfers were not different whether the embryos were fresh or fiozen (Table 1). Likewise, the pregnancy rate in cows was not different between surgical and non-surgical transfers of fresh embryos. Cows exhibited a lower pregnancy rate, however, when fiozcn embryos were transferred non-surgically compared to surgically. The pregnancy rate for cows was lower than for heifers for fresh embryos transferred either surgical or non-surgical, but not for tkozen embryos.
Table 1. Pregnancy rates following surgical or nonsurgical transfer of fresh or cozen-thawed embryos to cows or heifers. Fresh Frozen No. Transfers No. Transfers % Pregnant % Pregnant Surgical Heifers 1485 586 70.8’ 79.9a cows 90 491 69.7b 71.1’ Nonsurgical Heifers 590 78.8“ 72 59.7 cows 84 60.7b 26 38.5d 4b*c*dValuesin columns without common superscripts differ significantly (a vs b: P
Of the transfers in Table 1, 580 were performed on one fkm during a period of approximately one year. The mean quality grade of the 213 embryos transferred surgically was 1.61, compared to 1.62 for 367 embryos that were transferred nonsurgically. There was no significant difference between the 85.0% surgical and 79.3% non-surgical pregnancy rates on this farm
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Study 2 The overall pregnancy rate resulting from transfer of 9023 fresh embryos was 68.3%, compared to 58.4% (P
Table 2. The effect of geographic location, year and season on pregnancy rates of fresh and frozen-thawed embryos Fresh Frozen No. Transfers % Pregnant No. Transfers % Pregnant Year 1987 2512 68.6 1174 61.3 1988 2716 67.3 1290 58.5 1991 1960 70.1 1479 57.5 1992 1835 67.2 1354 56.7 Location EmT-W 5345 73.2a 2879 61.5’ EmT- 1 1173 62.2b 707 56.0 EmT-2 2505 61.0b 1711 54.1b Season Spring 2718 68.8 1394 58.2 Summer 2004 67.7 1691 60.9 Fall 1907 67.8 883 58.9 Winter 2394 68.5 55.1 1329 “,bValues in columns without common superscripts differ significantly (PcO.05)
There was no difference in pregnancy rates between embryos of dairy and beef origin whether they were transferred fresh or Cozen-thawed (Table 3). The pregnancy rate of dairy cowrecipients was lower (P
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effect of synchrony on the pregnancy rate of t?esh but not frozen-thawed embryos. embryos, the linear and quadratic effects significantly differed from zero (PcO.05).
For fresh
Table 3. The effect of embryo breed and recipient breed and parity on pregnancy rates of fresh and frozen-thawed embryos Fresh Frozen % Pregnant No. Transfers % Pregnant No. Transfers Donors Dairy 7457 68.5 4038 58.8 Beef 1566 67.3 1259 57.2 Recipients Dairy Heifers 6612 70.58 3477 60.9” Dairy Cows 844 52.gb 518 47.1b Beef Heifers 267 65.9’ 252 60.0’ Beef Cows 835 68.68 461 58.6” “Sbvalues in columns without common superscripts differ significantly (P
-e
Fresh
90 = 80 c” 70 p 80 a 50 $ 40 30 48
38
24 Plus
12
0
Synchrony
-12 (hours)
-24
-38
48
Minus
Figure 1. Effect of recipient-donor estrus synchrony on pregnancy rate following transfer of fresh and frozen-thawed embryos. Plus asynchrony=recipient in estrus before donor; Minus asynchrony=recipient in estrus after donor. Left to right, n for fksh embryos: 54, 149,979, 1836,3524, 1377,922, 142 and 13; for frozen embryos: 11, 125, 707, 1298,2124,753,227 and 26. The relationship between estrus synchrony and pregnancy rate following transfer of both fresh and frozen embryos into dairy heifers and cows and beef cows is shown in Figure 2. Although the pregnancy rate was lower in dairy cows than in beef cows or dairy heifers, the curves were very similar.
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I
q-Dairy
-
heifers
:r
48
Plus
36
24
+Beefcows
12
0
*Dairy
-12
Synchrony (hours)
-24
cows
-36
I
-48
Minus
Figure 2. Effect of recipient-donor estrus synchrony on pregnancy rate following transfer of fresh and frozen-thawed embryos (combined totals) into dairy heifers and cows and beef cows. Plus asynchrony = recipient in estrus before donor; h4inus asynchrony= recipient in estrus after donor. Left to right, n for dairy heifers: 40, 182, 1224,2240, 4026, 1434, 804, and 108; for dii cows: 137, 252, 548, 233 and 127; for beef cows: 129,298,490,236 and 103.
There were no differences in pregnancy rates of embryos frozen in 0.5 mL versus 0.25 mL straws (Table 4). Pregnancy rates were lower, however, following transfer of embryos that were frozen in glass ampules, than embryos frozen in either 0.5 or 0.25 mL straws.
Table 4. The effect of straw size and glass ampules on the pregnancy rate of recipients. No. Transfers % Pregnant Embryo container 0.5 mL straws 3245 58.9” 0.25 mL straws 2056 57.5a Glass ampules 62 30.6b “%alues in columns without common superscripts differ significantly (P
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Table 5. Effects of tksh and frozen-thawed embryo grade, stage of development and age on pregnancy of recipients Frozen Fresh % Pregnant No. Transfers % Pregnant No. Transfers Embrvo Grade 1 2 3
4163 3156 1641
73.2’ 68.3b 56.3’
2482 2329 454
61
47.5c
22
62.8’ 56.8b 43.6’
4 Embrvo Stage M EB MB xl3 HB
36.4b,”
5633 1978 995 391 25
66.9 70.3 70.9 71.4 56.0
3576 1140 478 93 __^
57.7 61.2 57.9 50.5 ---
Embrvo Ane (dl 5 5.5 6 6.5 7 7.5 8 8.5 9
13 13 225 490 6810 1051 339 36 23
76.9 61.5 65.8 70.0 68.8 65.4 67.6 72.2 34.8
_-_ --18 84 3794 1224 129 -___-
___ --_ 55.6 61.9 58.3 60.0 50.4 --___
a,b*cgdValues in columns without common superscripts differ significantly (P~0.05)
Study 3 Between 1988 to 1995, a total of 3616 embryos frozen at EmT were thawed and transferred by personnel at HG, resulting in 2030 pregnancies (56.1%). The location of transfers was divided between recipients owned and housed by HG (n-3 122) and recipients on private farms in The Netherlands (t&94). Analysis of variance showed that the following factors did not significantly affect pregnancy rate: year or season of Ikeezing, year or season of transfer, location of transfer, parity of recipient and transfer team. Likewise, the time between flushing and freezing did not affect pregnancy rate (Table 6). In addition, interactions were not significant (P
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Table 6. Effects of day of recipient estrous cycle, embryo grade and stage, and the time from flushing to lkeezing on pregnancy rate of frozen-thawed embryos No. Transfers % Pregnant Day of Recipient Estrous Cycle 53.6b 8 (plus)’ 774 59.0” 7 (0) 2015 48.9b 6 (minus) 468 Embryo Grade2 57.8’ 1 1723 54.3c 2 788 37.9* 3 253 Embryo Stage 54.3f M 1997 59.7e EB 494 52.1’ MB 257 Time Between Flushing and Freezing (min) 54.1 0 -30 85 56.0 30 -60 418 56.0 61-90 722 56.2 91-120 507 53.0 121-150 264 55.7 151-180 106 ‘Estrous synchrony, 2Before freezing, ‘*b~c~4e%aluesin columns without common superscripts differ significantly (a vs b: P
DISCUSSION During the early years of the ET industry, it was reported that pregnancy rates from NS transfer were lower than those from surgical transfer (20,28,31). One subsequent report also showed a higher pregnancy rate resulting tiom surgical transfer (33), while others reported no difference (2,lO). The present study indicated that comparable pregnancy rates can be achieved in heifers with both methods of transfer. Nevertheless, the data suggest that pregnancy rates may favor surgical transfers in cows. The 8.4 and 9.9 percentage point differences between the pregnancy rates of hesh and tiozen embryos in Studies 1 and 2 are similar to the range of differences for tiesh and t?ozen embryos (8.5 to 14) previously reported (8,15,19,30,32). In Study 2, there were similar differences in pregnancy rates between t+esh and frozen embryos at each grade level. This suggests that the decrease in pregnancy rate associated with frozen-thawed embryos is due to damage that is not visually obvious based on quality grading procedures.
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Data from Study 1 indicate that higher pregnancy rates were achieved in heifers than cows. Approximately 85% of the animals in this study were Holsteins, while the balance were a mixture of beef breeds. Similarly, in Study 2, transfer of either fresh or frozen embryos resulted in higher pregnancy rates in dairy heifers and in both beef heifers and cows than in dairy cows. Other investigators have also reported a higher pregnancy rate in dairy heifers than in dairy cows following NS transfer of either fresh (8) or frozen embryos (8,ll) whereas still others either reported no difference (7), or equivocal results (5) that were attributed to nutritional deficiencies in the cows. The differences between daii heifers and cows in the present study may be due to the higher stress levels associated with lactation that are imposed upon cows in many dairy facilities. It has been well documented that reproductive efficiency in dairy cattle has declined over the last several decades (6,27). Conception rate declined in dairy cows in the USA from 66% in 1951 to 50% in 1973, while average milk production increased by 33% (6). It is noteworthy that during this period, conception rate in virgin dairy heifers did not decline during this period and, if fact, heifers and cows were equally fertile to AI in 1954 (6). This suggests that the decline in cows is related to the stress of greater milk production and is not due to genetic selection for lower fertility. Nevertheless, the decline in fertility appears to be associated with a rise in genetic selection for milk production (18,23). Pryce et al. (23) reported that body condition score recorded 1 month after calving had the largest genetic correlation with calving interval. Modifications in diet may provide the greatest aid in counteracting the decrease in fertility that is related to genetic selection for increased milk production (3,23). Obviously, nutritional and lactational stress can also be of considerable importance in postpartum beef cows. However, neither the present study nor the study by Wright (36) showed any difference in pregnancy rates between beef heifers and cows, The lack of yearly differences in pregnancy rates of fresh or frozen embryos in the present study is consistent with some previous reports (9,ll). However, Hasler et al. (13) reported some yearly differences in pregnancy rate following surgical transfer into Holstein heifers over a 7-year period. In the present study, the interaction between year and location has no obvious biological explanation. It is somewhat surprising that there was no influence of season within location on pregnancy rate, considering the contrast in climate represented by central California and the northeast United States. However, other studies that included embryo transfers in Japan and in both the central and southwest United States, also failed to show any seasonal infhtences on pregnancy rate (9,11,24). Furthermore, Putney et al. (24) did not detect an influence of season on pregnancy rates following embryo transfas in the Southwest USA even though monthly mean environmental temperatures ranged Tom 7.8 to 28.6”C. The study by Lindner and Wright (16) is frequently used as a reference for embryo quality relative to pregnancy rate. Subsequently, the IETS published a grading system for bovine embryos (26) in which the separate excellent and good categories of Lindner and Wright (16) were combined into a single grade designated as number 1. Similarly, IETS Grade 2 embryos represent Lindner and Wright’s third category of fair embryos. The rating system in the present study more closely resembles that used by Lindner and Wright (16). Consistent criteria have not been employed for grading the morphological quality of cattle embryos in the literature (9,10,13,16,26,30,36). Nevertheless, the majority of these studies indicated significant differences in pregnancy rate among different grades of either frozen and/or &esh embryos.
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However, most studies have combined excellent and good embryos into one grade and, as a consequence not been able to relate the two grades to pregnancy rates. The present study is both supported (10,13) and contradicted (16,21,30) by several other studies in demonstrating differences in pregnancy rates between excellent and good quality embryos. Although the IETS combines the two grades into one for the purposes of international standardization, there may be situations where there are economic advantages to handle the two grades separately. Assessment of cattle embryo developmental stages is not subject to as much variation as is quality grading. Many studies have not involved sufficiently large sample sizes to detect small differences among stages. Nevertheless, in contrast to the present study, a number of studies have shown the transfer of t?esh blastocysts generally resulted in higher pregnancy rates than morulae (10,13,24,28,36). Other published reports have shown a similar pregnancy rate for morulae and blastocysts (21), a lower pregnancy rate for mid- and expanded-blastocysts than morulae (1 l), or no differences (35). Synchrony between the age of the embryo and the estrous cycle of the recipient has long received attention as potentially affecting the success rate of ET. The present study indicated differences (PcO.05) in pregnancy rates relative to estrous synchrony based on ANOVA. It is very clear, however, that for practical application in the field, 24 h plus or minus asynchrony resulted in pregnancy rates comparable to zero synchrony with either fresh or frozen-thawed embryos. There is no indication that frozen-thawed embryos are any more or less sensitive to asynchrony than are fresh embryos. Some previous studies on synchrony with fresh embryos have indicated that beef recipients were apparently more sensitive to asynchrony (10,24,28,35) than dairy heifers (9,14). There were no differences in pregnancy rate relative to asynchrony in the present study among beef cows, dairy cows and daii heifers. With rare exceptions (17), the initial successes with commercial freezing of cattle embryos involved the use of glass ampules (22,29). As pointed out in a recent review (25), however, higher rates of zona fkacture were associated with the use of glass ampules compared to plastic straws. Plastic straws have largely replaced glass ampules as the container of choice for freezing cattle embryos. The results of the present study show that there is no advantage of the size of the straw on embryo survival, but both sizes of straws resulted in higher pregnancy rates than glass ampules. The time between embryo collection and keezing is an important practical consideration in commercial ET. Previous studies (22,36) have shown that there was a drop in pregnancy rate when embryos were held for more than 4 h prior to freezing. Otter (21) reported that pregnancy rate decreased for embryos held for between 2 to 4 or 4 to 6 h, compared to 0 to 2 h prior to lkeezing. The present study showed no effect of time to hezing at intervals of 30 min up to 3 h following embryo collection. Virtually all the &zing, however, was conducted in a laboratory with an ambient temperature that was maintained below 28OC in summer. It is possible that maintainmg embryos at higher temperatures, such as those encountered in on-farm situations in summer would result in lower embryo survival rates. The cryoprotectant used for fkezing embryos in the present study was 10% glycerol, whereas 1.5 M ethylene glycol has become the predominant cryoprotectant in the commercial ET
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industry in the last few years. Published reports have indicated that survival is similar for both in vitro- (14) and in vivo-derived embryos (1,8,11,15) frozen in either of the two cryoprotectants. Consequently, it is reasonable to assume that the influence on pregnancy rate of the factors examined in this study will also apply to embryos frozen in ethylene glycol.
CONCLUSIONS There was no evident loss of pregnancy rate with the transition Iiom surgical to nonsurgical transfer of embryos in a commercial ET program Location and year, but not season affected pregnancy rate. There was a loss in pregnancy rate of approximately 10 to 13 percentage points between fresh and frozen-thawed embryos of comparable grades. Dairy cows sustained a lower pregnancy rate than daii heifers or beef heifers or cows. Although embryo quality was of paramount importance in determining pregnancy rate for both fresh and frozen-thawed embryos, embryo stage of development did not influence pregnancy rate. Estrous asynchrony affected fresh and frozen-thawed embryos pregnancy rates in a similar fashion, with no evident affect when synchrony was within 24 h plus or minus of zero.
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