Factors affecting success of embryo collection and transfer in large dairy herds

Available online at www.sciencedirect.com Theriogenology 69 (2008) 98 106 www.theriojournal.com Factors affecting success of embryo collection and transfer in large dairy herds R.C. Chebel a, *, D.G.B. Demétrio b, J. Metzger c a Veterinary Medicine Cooperative Extension, University of California Davis, 18830 Road 112, Tulare, CA 93274, USA b RuAnn and Maddox Dairy, 7285 W Davis Ave, Riverdale, CA 93656, USA c TransOva Genetics, 2938 380th Street, Sioux Center, IA 51250, USA Abstract Our objective was to evaluate factors that affected the success of embryo transfer programs in large dairy herds. Non-lactating donor cows produced a larger number of ova/embryos (P < 0.01) and viable embryos (P < 0.01) than lactating cows. The interaction between season and donor class was correlated with the proportion of ova/embryos classified as fertilized (P = 0.03), because lactating donors had fewer fertilized ova in the summer. There was no correlation between 305-day mature equivalent milk yield and response to superstimulation. Although the interval between superstimulation protocols was correlated with the number of ova/embryos (P = 0.03), there was no correlation with the number of viable embryos. Pregnancy per embryo transfer (P/ET) in heifer recipients was correlated with embryo quality grade (P < 0.01), season (P = 0.04), and whether embryos were fresh or frozen/thawed (P < 0.01). Lactating recipient cows tended to have a lower rate of P/ET during the summer (P = 0.12 to P = 0.08). Synchronization protocols tended to be (P = 0.06; Herd 1) or were (P = 0.02; Herd 2) correlated with P/ET. Lactating cows receiving vitrified IVF embryos had a lower (P = 0.01) P/ET than those receiving fresh IVF embryos, especially in the summer (P = 0.09). Milk yield was not correlated with P/ET. The use of heat abatement systems is critical to improve embryo production and P/ET. Synchronization protocols that optimized synchrony of ovulation may increase fertility of recipient cows and eliminate the need for estrous detection. # 2007 Published by Elsevier Inc. Keywords: Dairy cows; Embryo transfer; Heat stress; IVF; Superovulation 1. Introduction Approximately 5% of animals registered in the American Holstein Association Inc. in the 1990s were derived from embryo transfer [1]. The utilization of embryo transfer as a routine practice in reproductive management of large dairy herds in Western states of the USA is still limited. This is intriguing, because utilization of embryo transfer in commercial herds has * Corresponding author at: 18830 Road 112, Tulare, CA 93274, USA. Tel.: +1 559 688 1731; fax: +1 559 686 4231. E-mail address: rchebel@vmtrc.ucdavis.edu (R.C. Chebel). the potential to expedite genetic improvement, and consequently, improve production. Furthermore, the implementation of embryo transfer programs has the potential to mitigate the effects of heat stress on reproductive performance of lactating dairy cows [2,3]. The proportion of lactating dairy cows that become pregnant after AI has declined steadily in the past 50 years, and has been accompanied by a constant increase in milk yield [4]. In the past two decades, however, there has been little change in the average number of ova/ embryos and transferable embryos produced in embryo transfer programs in the USA and Canada [5]. Furthermore, there was no difference in the number of ova/embryos and viable embryos produced by cows 0093-691X/$ see front matter # 2007 Published by Elsevier Inc. doi:10.1016/j.theriogenology.2007.09.008

R.C. Chebel et al. / Theriogenology 69 (2008) 98 106 99 of different milk yield [5]. Although these findings were related to superovulated cows, this observation corroborated the findings of studies that have demonstrated no direct correlation between level of milk production and conception or pregnancy loss in lactating Holstein cows [6,7]. Furthermore, Hasler [5] reported little change in the proportion of recipient animals that become pregnant following embryo transfer, over a period of approximately 30 years. The constant increase in size of dairy operations has posed challenges to the management and individual care of lactating cows, and may be one of the factors contributing to the reduced reproductive efficiency in large dairy herds [4]. Our objective was to evaluate factors that may affect the success of on-farm embryo transfer programs in large dairy herds. 2. Materials and methods 2.1. Superovulation Data from an embryo transfer program on a dairy located in the San Joaquin Valley of California with 1100 lactating Holstein cows and a 305-day rolling herd average of 12,272 kg/year were used. Approximately 135 non-lactating embryo donors were housed in dry-lot corrals with shades, whereas lactating donors were housed in free-stall pens. There were no heat abatement systems in place. A total of 702 superovulations in 363 cows (lactating = 248, non-lactating = 115) were performed from July 2006 to June 2007. Donors were observed daily for signs of estrus, and superstimulatory treatments were initiated between 8 and 10 days after detected estrus. Cows received FSH twice a day, over a 4-day period, in decreasing doses. On the third day of FSH treatment, cows were given PGF2a at the same time as the fifth treatment of FSH; GnRH was administered 48 h after PGF2a, and cows were inseminated 12 and 24 h later. In cows with 2 CL, non-surgical embryo collections were done 7 days after the first AI. All embryos and ova collected were classified (by one technician) according to the guidelines of the International Embryo Transfer Society [7]. Number of ova/embryo, viable embryos, and unfertilized ova were recorded. 2.2. Synchronization and data collection for recipients 2.2.1. Recipient heifers California In two dairy farms located in the San Joaquin Valley of California, 1183 embryos were transferred to nulliparous heifers from September 2006 to June 2007. Embryo transfers were performed in the summer (May September) and winter (October April). At one of the sites, recipient heifers were housed in open corrals, whereas at the other site, heifers were housed in free-stall barns, but in both locations no cooling systems were used. Heifers were observed daily for signs of spontaneous estrus; those detected in estrus received an in vivo-produced fresh or frozen/thawed embryo 6, 7, or 8 days later. Embryos were frozen in ethylene glycol for direct transfer [9]. Embryos were classified according to developmental stage and quality grade [8]. Pregnancy was diagnosed at approximately 42 days of gestation. 2.2.2. Lactating dairy recipients Herd 1 had approximately 1500 lactating dairy cows during the study period (May 2005 January 2006). The majority of the cows were Holstein, but there were a few Holstein Jersey crossbred animals. Lactating dairy cows that were diagnosed not pregnant to a previous AI or embryo transfer received GnRH on Day 0, PGF2a on Day 7, and GnRH on Day 10 (Ovsynch protocol) [10], with the addition of a CIDR 1 (Pfizer Animal Health, New York, NY, USA) containing 1.38 g of progesterone from Day 0 to 7. Seven days after the last GnRH (Day 17), cows with a CL received an embryo. Therefore, cows were classified according to reproductive code as non-bred and re-bred. During the summer of 2005, cows received a fresh IVF embryo produced with sexed semen, and during the winter of 2005/2006, cows received an IVF embryo or a frozen/thawed in vivoproduced embryo cryopreserved in glycerol [9]. Herd 2, located in South Dakota, had approximately 900 lactating Holstein, Jersey, and crossbred dairy cows during the winter (February April) and summer (May June) of 2006. Cows that had not yet been inseminated after calving were presynchronized with PGF2a, and the Ovsynch protocol with a CIDR was initiated 14 days after the last PGF2a. Cows that had been inseminated and diagnosed not pregnant were resynchronized with either the Ovsynch protocol plus CIDR or with the Ovsynch protocol alone. Seven days after the last GnRH, all cows with a CL received either a fresh or vitrified IVF embryo produced with sexed semen from Jersey sires and oocytes from Holstein dams. In vitro fertilized embryos were vitrified in 0.25 ml straws. Pregnancy was diagnosed at approximately 35 days of gestation. 2.3. Statistical analyses Continuous data were analyzed by ANOVA using the GLM procedure of SAS (Statistical Analysis Software,

100 R.C. Chebel et al. / Theriogenology 69 (2008) 98 106 Table 1 Correlations between classes of donors and season and mean (S.E.M.) numbers of ova/embryos and viable embryos and the proportion of fertilized ova for all cows superstimulated between July 2006 and June 2007 Item Donor class P values Non-lactating Lactating Donor Season Donor season Summer a Winter a Summer a Winter a No. 144 200 87 221 Ova/embryos 13.8 1.0 14.5 0.9 6.6 1.2 7.8 0.9 <0.01 0.31 0.85 Fertilized (%) 51.0 4.6 49.1 4.3 41.8 5.6 a 56.7 4.3 b 0.87 0.12 0.03 Viable embryos 5.4 0.7 5.0 0.7 1.7 0.8 a 3.4 0.6 b <0.01 0.31 0.08 (a, b) Within donor class, means with different superscript differ (P < 0.05). a Seasons. SAS Institute Inc., Cary, NC, USA). The independent variables included in the models were season, class of donor cow, brand of FSH used, 305ME, service sire, and superstimulations intervals (once, <60 days interval, 60 days interval). Dichotomous data were analyzed by logistic regression using the LOGISTIC procedure of SAS, using a backward stepwise multivariate logistic model with variables removed from the model by the Wald statistics criterion if the significance was >0.15. The model used for analysis of factors correlated with P/ET in recipient heifers included season, donor cow, donor class, sire, embryo quality grade, and developmental stage, type of embryo, site, estrus synchrony between recipient and donor cattle, and technician. The model used for analysis of factors correlated with the proportion of lactating cows selected to receive an embryo included season, breed, parity, DIM, milk yield, and synchronization protocol. The model for analysis of factors associated with P/ET included parity, synchronization protocol, type of embryos, milk yield, DIM, season, and technician. Reproductive code was included in the model for Herd 1, whereas individual cow linear somatic cell score in the test prior to embryo transfer in Herd 2 was included in the model. The adjusted odds ratio estimates and the 95% confidence intervals from the logistic regression were obtained for each variable. The regression procedure of MINITAB (MINITAB 1, MINITAB Inc., State College, PA, USA) was used to determine the fitted line plot that best described the relationship between the adjusted odds ratio estimates and P/ET and linear somatic cell scores. 3. Results 3.1. Factors affecting response to superovulation From the 702 cows superstimulated, 652 (92.9%) had 2 CL on the day of embryo recovery. A larger proportion of non-lactating than lactating cows was selected for embryo recovery (98.9 vs. 87.0%; P < 0.01). There was a tendency (P < 0.15) for brand of FSH and season (summer = 91.7%, winter = 93.6%) to be correlated with the proportion of donor cows bearing 2 CL. Interval between superstimulations was not (P = 0.4) correlated with the proportion of cows selected for embryo recovery. A mean of 10.9 0.4 ova/embryos was collected; this was correlated with the class of donors (P < 0.01; Table 1) and with the interval between superstimulations (once = 11.0 0.7, <60 days = 8.6 1.2, 60 days = 12.4 0.9; P = 0.03). The brand of FSH was correlated (P = 0.02) with the number of ova/embryos. Season (P = 0.31) and 305ME (P = 0.7) were not correlated with the number of ova/embryos. The proportion of ova/embryos classified as fertilized tended (P = 0.1) to be correlated with season, but not with donor class (P = 0.9; Table 1). However, the interaction between season and donor class was correlated (P = 0.03) with the proportion of fertilized ova. The proportion of ova/embryos classified as fertilized tended to be correlated with interval between superstimulations (P = 0.1; once = 52.2 3.5, <60 days = 53.6 5.3, 60 days = 43.1 4.2%), and was correlated with AI sire (P < 0.01), but not with brand of FSH (P = 0.9) or 305ME (P = 0.2). Ameanof4.7 0.2 viable embryos were collected, which represented 83.5% of all fertilized ova/embryos. The mean number of viable embryos was correlated (P < 0.01) with the donor class, but not with season (P = 0.3); the interaction between donor class and season tended to be correlated mean number of viable embryos (P =0.08; Table 1). The interval between superstimulations (P = 0.9), the brand of FSH (P =0.5), milkyield (P =0.4), and AI sire (P =0.5) were not correlated with the mean number of viable embryos.

R.C. Chebel et al. / Theriogenology 69 (2008) 98 106 101 Table 2 Description of factors correlated with the proportion of heifers that become pregnant following embryo transfer (P/ET) Items P/ET (no./no.) P value Embryo quality grade 1 59.4 (443/746) <0.01 2 53.8 (157/292) 3 35.2 (51/145) Donor class Non-lactating 52.2 (405/776) 0.83 Lactating 60.4 (246/407) Embryo status Frozen/thawed 44.2 (76/172) <0.01 Fresh 56.9 (575/1011) Synchronization (days) 1 58.6 (51/87) 0.99 0 53.7 (464/864) +1 58.6 (136/232) Season Summer 52.0 (120/231) 0.04 Winter 55.8 (531/952) 3.2. Factors correlated with synchronization and pregnancy per embryo transfer A total of 651 heifers in California were diagnosed pregnant after embryo transfer (55.0% of all the heifers receiving an embryo). The factors correlated with P/ET were embryo quality grade (P < 0.01), type of embryo (P < 0.01), season (P = 0.04), and technician (P < 0.01; Table 2). Embryo developmental stage (P = 0.4), donor (P = 0.8), class of donor (P = 0.8), AI sire (P = 0.6), site (P = 0.9), and estrus synchrony (P = 0.99) were not correlated with P/ET. A total of 368 lactating cows South Dakota (Herd 1) were synchronized (n = 57) or resynchronized (n = 311), and 305 (82.9%) had a CL on the day of embryo transfer. There was no correlation with parity (P = 0.6), reproductive code (P = 0.9), season (P = 0.4), breed (P = 0.3), or DIM (P = 0.2; Table 3). However, milk yield was correlated (P = 0.05) with the proportion of cows bearing at least one CL (MQ1 = 81.4, MQ2 = 90.6, MQ3 = 75.0, MQ4 = 83.7%). Data from 298 embryo transfers were analyzed. The average milk yield was 36.0 0.4 and 32.3 0.7 kg/day for multiparous and primiparous recipients, respectively, and the average DIM was 180.0 4.5 days. There was a tendency for season to be correlated (P = 0.1) with P/ET, and for primiparous cows to have higher P/ET than multiparous cows (P = 0.1; Table 4). There was a tendency (P = 0.06) for reproductive code to be correlated with P/ET (non-bred = 36.5 and re-bred = 45.9%). Non-bred cows averaged 80.1 8.7 DIM, while re-bred cows averaged 201.2 4.0 DIM (P < 0.01). There was no correlation between type of embryo (P = 0.8), breed of recipient (P = 0.8), milk yield (P = 0.9), DIM (P = 0.4), or technician (P = 0.7) and P/ET. A total of 509 cows were synchronized in Herd 2 and 408 (80.2%) had a CL on the day of embryo transfer. Synchronization protocol (P = 0.03) and milk yield (P = 0.01) were correlated with the proportion of cows bearing a CL (MQ1 = 69.2, MQ2 = 84.7, MQ3 = 81.7, MQ4 = 84.1%). Breed of recipient tended (P = 0.1) to be correlated with the proportion of cows with a CL Table 3 Correlation among parity, season, and synchronization protocol and proportion of cows selected to receive an embryo (SR) in Herds 1 and 2 (South Dakota) Herd 1 Herd 2 SR (%) (no./no.) P value SR (%) (no./no.) P value Parity Primiparous 82.5 (80/97) 0.62 78.6 (158/201) 0.56 Multiparous 83.0 (225/271) 81.2 (250/308) Season Summer 82.5 (189/229) 0.43 79.6 (172/216) 0.71 Winter 83.5 (116/139) 80.6 (236/293) SP a POVC N/A 0.95 86.9 a (166/191) 0.03 ROVC N/A 77.5 b (155/200) ROV N/A 73.7 b (87/118) (a, b) Within item and herd, proportions with different superscripts differ (P < 0.05). a Synchronization programs: POVC Days 28 and 14: PGF2a; Day 0: GnRH and CIDR; Day 7: PGF2a and CIDR removal; Day 10: GnRH; ROVC Day 0: GnRH and CIDR; Day 7: PGF2a and CIDR removal; Day 10: GnRH, for re-bred cows; ROV: same as ROVC but without a CIDR.

102 R.C. Chebel et al. / Theriogenology 69 (2008) 98 106 Table 4 Correlation among parity, season, synchronization protocol, and type of embryo and proportion of cows diagnosed pregnant after embryo transfer (P/ET) in herds 1 and 2 Herd 1 Herd 2 P/ET (n/n) P value P/ET (n/n) P value Parity Primiparous 50.6 (40/79) 0.14 57.6 (91/158) 0.24 Multiparous 42.0 (92/219) 50.6 (125/247) Season Summer 42.1 (77/183) 0.12 47.7 (81/170) 0.08 Winter 47.8 (55/115) 57.5 (135/235) SP * POVC N/A 0.06 N/A 0.02 ROVC N/A 57.3 a (94/164) POVC N/A 57.1 a (88/154) ROV N/A 39.1 b (34/87) Type of embryo ** FIVF 44.3 (101/228) 0.81 56.8 (168/296) 0.01 FTIV 44.3 (31/70) N/A VIVF N/A 44.0 (48/109) a,b Proportions with different superscripts within Item and herd differ (P < 0.05). * Synchronization programs. POVC: d 28 and 14 PGF, d 0 GnRH and CIDR insertion, d 7 PGF and CIDR removal, d 10 GnRH; OVC: d 0 GnRH and CIDR insertion, d 7 PGF and CIDR removal, d 10 GnRH; ROVC: same as OVC for cows diagnosed non-pregnant to previous AI or EI; ROV: same as ROVC but without a CIDR. ** Type of embryo. FIVF: fresh IVF embryo; FTIV: frozen/thawed in vivo-produced embryo; VIVF: vitrified IVF embryo. (Holstein = 73.1, Jersey = 86.1, crossbred = 80.5%), and factors such as season (P = 0.7), parity (P = 0.6), and DIM (P = 0.5) were not correlated with the presence of a CL (Table 3). Pregnancy per embryo transfer was correlated (P = 0.02) with the synchronization protocol (Table 4). The average DIM differed (P < 0.01) among cows submitted to the different synchronization protocols (presynchronization = 79.8 4.6, Ovsynch = 176.5 6.3, and resynchronization = 182.9 4.8 days). The type of embryo was correlated (P = 0.01) with P/ET and there was a tendency (P = 0.08) for season and an interaction (P = 0.09) between season and type of embryo to be correlated with P/ET; cows receiving fresh IVF embryos had similar (P > 0.15) P/ ET in summer (55.4%) and winter (57.2%), while cows receiving vitrified IVF embryos tended (P = 0.09) to have a lower P/ET in the summer (41.7%) than during the winter (61.5%). Technician was (P < 0.01) and DIM (DIMQ1 = 64.5, DIMQ2 = 50.4, DIMQ3 = 47.1, DIMQ4 = 55.0%) tended (P = 0.07) to be correlated with P/ET. Linear somatic cell score (LSCL) also tended (P = 0.08) to be correlated with P/ET in Herd 2; as LSCL increased by 1 unit, the likelihood of pregnancy was reduced by 6.9%. Parity (P = 0.2), breed of recipient (P = 0.9), and milk yield (P = 0.2) were not correlated with P/ET. 4. Discussion The mean number of ova/embryos recovered in the current study was within the range reported by Hasler [5]. Interestingly, non-lactating dairy cows had a greater number of ova/embryos than lactating cows. Although some have not reported a difference between lactating dairy cows and non-lactating dairy cows [5] or nulliparous dairy heifers [11], lactating dairy cows have been reported to produce fewer ova/embryos than beef cows [12]. However, brand and dose of FSH and size of animals may have accounted for these differences. Lactating cows also had a smaller proportion of fertilized ova than non-lactating dairy cows. Although Sartori et al. [13] did not detect a difference in the proportion of fertilized ova from single-ovulating lactating and non-lactating dairy cows, superstimulated dairy cows produced fewer fertilized ova/embryos than beef cows [12]. An interaction between class of donor and season was detected in the present study. Sartori et al. [13] also reported that single-ovulating nulliparous heifers produced a larger proportion of fertilized ova than lactating dairy cows during the summer. The greater dry matter intake of lactating dairy cows compared to non-lactating dairy cows has been reported to increase the metabolism of estradiol [14], which is essential for fertilization [15]. Furthermore, during the

R.C. Chebel et al. / Theriogenology 69 (2008) 98 106 103 summer, lactating dairy cows had higher body temperature than nulliparous heifers, whereas during the winter, lactating dairy cows had body temperatures approximately 0.2 8C higher than non-lactating cows [13]. Exposure to heat and increased body temperatures have been linked to degeneration of thecal and granulosa cells, lower quality oocytes, and smaller fertilization and conception rates [6,13,16,17]. The mean number of viable embryos was smaller for lactating dairy cows compared to non-lactating cows, in the present study. Hasler [5], however, observed an opposite effect, with non-lactating cows had fewer viable embryos than lactating dairy cows. Putney et al. [12] observed that beef cows produced more viable embryos than lactating dairy cows, and that single-ovulating lactating dairy cows produced a larger proportion of viable embryos than non-lactating dairy cows. The changes in metabolism associated with lactation seem to be associated with reduced fertility by multiple pathways. Reduced concentrations of estradiol and progesterone associated with increased dry matter intake may result in follicle persistence, reducing the quality of oocytes [18] and compromising embryo development [19]. In addition, the increased feed intake associated with lactation resulted in increased heat production and reduced capability of thermoregulation in lactating dairy cows [20,21]. In the present study, only lactating dairy cows yielded fewer viable embryos during the summer than during the winter. There was no correlation between 305ME and response to the superstimulatory treatment for lactating and non-lactating dairy cows, whereas Hasler [5] reported that donor cows with milk yield >41 kg/day produced more viable embryos than non-lactating. The continuing increase in milk yield has been suggested to contribute to the decreased fertility in lactating dairy cows [4]. However, recent studies have not been able to determine a direct relationship between increased milk yield and decreased reproductive performance in large dairy herds [6,7]. Interval between superstimulations was not correlated with the number of viable embryos recovered. Lucy et al. [22] reported that treatment of donor cows with PGF2a at different intervals following embryo recovery resulted in resumption of normal cyclicity shortly thereafter. Therefore, the treatment of donor cows with PGF2a immediately after embryo collection caused resumption of normal ovarian cyclicity and allowed for initiation of superstimulation shortly after embryo collection, without compromising embryo quality. The fertility of nulliparous dairy heifers that received fresh in vivo-produced embryos was similar to that reported for dairy heifers receiving AI [23,24], and slightly lower than heifers receiving in vivo-produced embryos [5]. The P/ET after receiving a frozen/thawed embryo, however, was considerably smaller than that reported previously [25]. Ethylene glycol was used in the present study, whereas glycerol was used in other studies [25]. Although embryos frozen in ethylene glycol were transferred directly after thawing, those frozen in glycerol were evaluated and selected prior to transfer. The lower P/ET in heifers receiving frozen/ thawed embryos as compared to fresh embryos was consistent with other studies [25,26]. In the current study, quality grade of embryos was correlated with P/ET. Although Hasler [25] also detected differences in P/ET according to quality grade of embryos, there was no difference in beef recipients [26]. Developmental stage, which was less subjective than quality grade, was not correlated with P/ET in this study, which was corroborated by two experiments described by Hasler [25] and one described by Spell et al. [26]. Recipient heifers receiving early blastocysts, however, had consistently higher P/ET than those receiving morulas [25,27 29]. The lack of a correlation between estrus synchrony in donors and recipients and P/ET was corroborated by Spell et al. [26]; they did not observe differences when beef recipient cows received embryos at 24, 12, 0, +12, or +24 h synchrony to the donor cows. Although Hasler [25] observed a difference in P/ET for recipients receiving fresh embryos according to estrus synchrony, it was clear that the minimal difference between 24 and +24 h compared to the 0 h synchrony was not relevant in a commercial setting. One of the intriguing findings in this study was that recipient dairy heifers receiving embryos during the summer had significantly lower P/ET than heifers receiving embryos during the winter. Although it is generally accepted that dairy heifers are less sensitive to heat stress than lactating cows [13], heat stress may also reduce fertility in heifers. Donovan et al. [30] reported a decrease in conception rates of 31 percentage units in heifers inseminated during the summer compared to the winter in Florida. Furthermore, superovulated heifers produced more poor and degenerated embryos during exposure to heat stress [12,29]. Therefore, measures to mitigate the effects of heat stress on fertility of recipient heifers are warranted and should increase P/ET. The proportion of lactating cows selected to receive an embryo was similar in Herds 1 and 2. A greater proportion of cows submitted to the presynchronization protocol received an embryo than those submitted to the resynchronization with an Osynch protocol, with or

104 R.C. Chebel et al. / Theriogenology 69 (2008) 98 106 without the addition of a CIDR. The proportion of cows ovulating in response to the first GnRH was correlated with the stage of the estrous cycle when it is initiated [31,32]. Presynchronization of cows with two injections of PGF2a, with the second given 12 days before the initiation of the Ovsynch, assured that a greater proportion of cows ovulated in response to the first GnRH [33,34]. Although the use of CIDR inhibited the occurrence of estrus and ovulation in cows with spontaneous luteolysis [35,36], others reported no difference in the proportion of recipients receiving embryos following Ovsynch versus Ovsynch + progestin protocols [37]. The overall P/ET also in Herds 1 and 2 was 49.5%. Although this was higher than reported in some studies [2,3], it was similar to that reported by others that have used fresh [25,38,39] and frozen/thawed [25] in vivoproduced embryos or fresh and frozen/thawed IVF embryos [40]. There was no difference in P/ET for cows receiving in vitro-produced embryos and those receiving frozen/thawed in vivo-produced embryos in Herd 1. Xu et al. [40] obtained similar P/ET in recipients receiving vitrified IVF embryos from sexed and unsexed semen, and from frozen/thawed in vivo-produced embryos. The findings of the current study, however, were interesting because frozen/thawed in vivo-produced embryos were only transferred during the winter, which was expected to increase P/ET. In Herd 2, fresh IVF embryos resulted in greater P/ET than vitrified IVF embryos. Al-Katanani et al. [3] also reported greater P/ ET in lactating dairy cows that received fresh IVF embryos than vitrified IVF embryos. Lactating cows that received embryos during the summer tended to have smaller P/ET in both herds. Although Hasler [25] did not detect an effect of season on P/ET, recent studies have demonstrated a negative correlation between body temperature on the day of embryo transfer and P/ET in lactating dairy cows exposed to heat stress [38,39]. In Herd 2, heat stress decreased P/ ET in cows receiving vitrified IVF embryos, but not fresh IVF embryos. Ambrose et al. [2] and Al-Katanani et al. [3] also observed smaller P/ET in lactating dairy cows exposed to heat stress that received vitrified IVF embryos compared to fresh IVF embryos; exposure of recipients to heat stress is a further stressor on embryos that have undergone cryopreservation. Cows in Herd 2 that were submitted to the presynchronization and resynchronization protocol with a CIDR had greater P/ET than those resynchronized without a CIDR. The presynchronization of lactating cows with two PGF2a treatments, with the second given 12 or 14 days prior to the initiation of the Ovsynch, increased conception rates in lactating dairy cows following AI [33,34]. Because CIDR inhibited the occurrence of ovulation in cows that have spontaneous luteolysis, synchrony of estrus and ovulation were improved after CIDR removal [35,36,41]. Interestingly, non-bred cows submitted to the Ovsynch + CIDR protocol in Herd 1 had smaller P/ET than re-bred cows submitted to the Ovsynch + CIDR protocol. Re-bred cows had greater DIM, which may have improved results; the incidence of the anovular condition early postpartum was increased and correlated with lower fertility [41]. Linear somatic cell score tended to be negatively correlated with P/ET. Previous studies have clearly demonstrated a strong negative correlation between occurrence of clinical [6,42] and subclinical [43] mastitis and fertility in lactating dairy cows that were inseminated; therefore, mastitis may be associated with early embryonic death. 5. Conclusions It is important to note that the current data were retrospectively derived; therefore, it is only possible to establish correlations between independent and dependent variables. Nonetheless, the efficiency of embryo transfer programs in large dairy herds was affected by a number of animal, managerial, and environmental conditions. Response to the superstimulatory treatment was less in lactating dairy cows, which was further compromised by exposure to heat stress, indicating that providing efficient heat abatement would be expected to improve the number of viable embryos produced. Interestingly, there was no correlation between milk yield and number of viable embryos, suggesting that factors associated with lactation other than level of milk yield may affect fertility. Although quality grade classification of embryos was subjective, embryos of lower quality yielded a lower P/ET rate. Exposure of recipient nulliparous heifers and lactating dairy recipients to heat stress was associated with a reduced P/ET, indicating that managerial measures to reduce heat stress have the potential to increase pregnancy rates in large dairy herds. Finally, synchronization protocols that optimize synchrony of ovulation should result in acceptable reproductive performance in lactating dairy recipients. References [1] Hasler JF. The current status and future of commercial embryo transfer in cattle. Anim Reprod Sci 2001;79:245 64.

R.C. Chebel et al. / Theriogenology 69 (2008) 98 106 105 [2] Ambrose JD, Drost M, Monson RL, Rutledge JJ, Leibfried- Rutledge ML, Thatcher MJ, et al. Efficacy of timed embryo transfer with fresh and frozen in vitro produced embryos to increase pregnancy rates in heat-stressed dairy cattle. J Dairy Sci 1999;82:2369 76. [3] Al-Katanani YM, Paula-Lopes FF, Hansen PJ. Effect of season and exposure to heat stress on oocyte competence in Holstein cows. J Dairy Sci 2002;85:390 6. [4] Lucy MC. Reproductive loss in high-producing dairy cattle: where will it end? J Dairy Sci 2001;84:1277 93. [5] Hasler JF. The Holstein cow in embryo transfer today as compared to 20 years ago. Theriogenology 2006;65:4 16. [6] Chebel RC, Santos JE, Reynolds JP, Cerri RL, Juchem SO, Overton M. Factors affecting conception rate after artificial insemination and pregnancy loss in lactating dairy cows. Anim Reprod Sci 2004;84:239 55. [7] Santos JEP, Rutigliano HM, Sá Filho MF. 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