Palpable Corpus Luteum Research Articles

Dynamics of hypotrophic changes in the morphological formations of the ovaries of heifers of mating age on the 7-9th day of luteogenesis

Real-time in vivo studies of the physiological or pathological state of the ovaries of cows and heifers are currently relevant because it is the morphological and functional state of the gonads of female cattle that directly affects the effectiveness of biotechnical procedures used in practice: fertilisation, stimulation, and synchronisation of sexual function. The purpose of this study was to find the quantitative and qualitative indicators of the morphological and functional state of the ovaries of Ukrainian red dairy heifers during the economic year under loose housing conditions of industrial dairy complex. The study used a modified differential diagnosis of the morphometric and functional state of the ovaries at a fixed time of the sexual cycle – from 7 to 9 days after ovulation of the dominant follicle (Day 0 of the cycle). To compare the results of transrectal examination of heifers in different seasons of the year, the technique of visualisation of palpation data in vivo was used. 175 heads of heifers of mating age were examined, among which on the 7-9th day of the sexual cycle, 55.42% were found to have a palpable corpus luteum in the ovary according to the stage of development, 26.96% had premature lysis of the corpus luteum, 8.57% had morphological and functional formations on the surface of the ovary, and 9.14% had symptoms of degenerative gonadal pathologies. Subsequently, animals with degenerative changes in the ovaries (follicular kytosis) did not take part in the study. The remaining heifers had ovaries with symptoms of indepth hypotrophic changes in the tissues (30.00%) or signs of premature lysis of the corpus luteum (33.33%). During the economic year, a pronounced dynamics in the growth or decrease of hypotrophic phenomena in ovarian tissues was noted; the best indicators of morphometry and functional activity of heifers were observed in the warm season, which indicated a considerable effect of fresh air and solar insolation on the sexual function of heifers of mating age. It was found that the level of fertility of heifers during the year (an average of 40.02%) indicated the presence of errors in the technology of growing replacement stock in an industrial enterprise. The findings can be useful for optimising the schemes of hormonal synchronisation of the sexual cycle of replacement heifers and for the correct selection of hormonal therapy in case of symptoms of decreased sexual activity

Comparison of the 7 & 7 Synch protocol and the 7-day CO-Synch + CIDR protocol among recipient beef cows in an embryo transfer program

An experiment was designed to evaluate the effectiveness of the recently developed 7 & 7 Synch protocol to synchronize estrus among recipients prior to embryo transfer (ET). Postpartum beef cows (n = 1358) across thirteen locations were assigned to either the 7-d CO-Synch + CIDR protocol or the 7 & 7 Synch protocol prior to ET. Cows were preassigned to balanced treatments within location based on age and days postpartum, with body condition score recorded at ET. Cows assigned to the 7-d CO-Synch + CIDR protocol were administered gonadotropin-releasing hormone (GnRH; 100 μg gonadorelin acetate) on Day 7, an intravaginal controlled internal drug release (CIDR; 1.38 g progesterone) from Day 7 to Day 14, and prostaglandin F2α (PGF2α; 25 mg dinoprost tromethamine) coincident with CIDR removal on Day 14. Cows assigned to the 7 & 7 Synch protocol were administered PGF2α (25 mg dinoprost tromethamine) coincident with CIDR insertion on Day 0, GnRH (100 μg gonadorelin acetate) on Day 7, and PGF2α (25 mg dinoprost tromethamine) coincident with CIDR removal on Day 14. Cows were observed for visible signs of estrus, with GnRH (100 μg gonadorelin acetate) administered to cows failing to express estrus during the detection period. Embryo transfer was performed approximately seven days after estrus or GnRH administration. Presence of corpora lutea (CL) was determined via transrectal palpation by a single veterinarian blinded to treatment, and embryos were transferred only to cows with palpable CL. Embryo transfer was performed using either fresh or frozen embryos, with embryo stage and grade recorded for each recipient. The proportion of cows expressing estrus was increased (P < 0.0001) among cows assigned to the 7 & 7 Synch protocol (86% [529/615] vs 76% [488/640]). The proportion of cows expressing estrus and presenting with palpable CL at ET was greater (P < 0.0001) among cows following treatment with the 7 & 7 Synch protocol compared to the 7-d CO-Synch + CIDR protocol (76% [466/615] vs 65% [418/640]). Consequently, the proportion pregnant to ET was greater (P < 0.03) following the 7 & 7 Synch protocol (40% [263/653]) compared to the 7-d CO-Synch + CIDR protocol (34% [228/664]). In summary, the 7 & 7 Synch protocol involving administration of PGF2α and treatment with a CIDR for 7 days prior to GnRH improved the likelihood of estrus expression in recipients, increased the proportion of cows eligible to receive an embryo, which resulted in a greater pregnancy rate to ET.

166 Comparison of the 7 and 7 Synch protocol and the 7-day CO-Synch + controlled internal drug release protocol among recipient beef cows in an embryo transfer programme

An experiment was designed to evaluate the effectiveness of the recently developed 7 and 7 Synch protocol to synchronise oestrus and ovulation among recipients before embryo transfer (ET). Postpartum beef cows (n=1358) across 13 locations were assigned to either the 7-day CO-Synch + controlled internal drug release (CIDR) protocol or the 7 and 7 Synch protocol before oestrus detection and subsequent ET. Cows were pre-assigned to balanced treatments within location based on age and days postpartum, and body condition score was recorded at ET. Cows assigned to the 7-day CO-Synch + CIDR protocol were administered gonadotrophin-releasing hormone (GnRH; 100µg of gonadorelin acetate) on Day 0, an intravaginal CIDR insert (1.38g of progesterone) from Day 0 to 7, and prostaglandin F2α (PG; 25mg of dinoprost tromethamine) coincident with CIDR removal on Day 7. Cows assigned to the 7 and 7 Synch protocol were administered PG (25mg of dinoprost tromethamine) coincident with CIDR insertion on Day −7, GnRH (100µg of gonadorelin acetate) on Day 0, and PG (25mg of dinoprost tromethamine) coincident with CIDR removal on Day 7. The 7 and 7 Synch protocol was hypothesised to enhance response to GnRH administration on Day 0 among mixed groups of oestrous cycling and anestrous cows, ultimately resulting in improved oestrous response and synchrony of oestrus before ET. Cows were observed for visible signs of oestrus following oestrus synchronisation, with GnRH (100µg of gonadorelin acetate) administered to cows failing to express oestrus during the detection period. Length of the detection period varied among locations but was consistent across treatments within location. Embryo transfer was performed approximately 7 days after oestrus or GnRH administration. Presence of corpora lutea (CL) was determined via transrectal palpation by a single veterinarian blinded to treatment, and embryos were transferred only to cows with palpable CL. Embryo transfer was performed using fresh or frozen embryos staged and graded according to IETS recommended guidelines, with embryo information recorded for each recipient. Data were analysed using the PROC GLIMMIX procedure of SAS (SAS Institute Inc.). Proportion of cows expressing oestrus was improved (P&amp;lt;0.0001) among cows assigned to the 7 and 7 Synch protocol (86% (529/615) vs. 76% (488/640)). The proportion of recipient females with palpable CL tended to be greater (P=0.07) among cows assigned to the 7 and 7 Synch protocol (82% (478/586) vs. 80% (451/565)). Among cows that expressed oestrus, a greater proportion (P&amp;lt;0.01) had palpable CL following the 7 and 7 Synch protocol (88% (466/529) vs. 86% (418/488)). Consequently, irrespective of embryo type received, the proportion of recipients pregnant to ET was greater (P&amp;lt;0.01) following the 7 and 7 Synch protocol (39%; 263/668) compared with the 7-day CO-Synch + CIDR protocol (33%; 228/690). In summary, the 7 and 7 Synch protocol improved the likelihood of oestrus expression in recipient cows, increased the proportion eligible to receive an embryo, and resulted in greater pregnancy rate to ET.

100 Relationship between corpus luteum blood flow evaluated via colour Doppler ultrasound and pregnancy rate in bovine embryo transfer recipients

The advent of colour Doppler ultrasound has allowed evaluation of blood flow to bodily tissues. This novel technology is being tested as a tool to improve efficiency in a variety of livestock reproduction programs. The objective of the present study was to evaluate the relationship between blood flow to the ovarian corpus luteum (CL), imaged via colour Doppler ultrasound, and pregnancy rate in crossbred dairy recipients for embryo transfer (ET). Oestrous cycles of Bos indicus×Bos taurus dairy heifers (n=90, 16-24 months of age) were synchronized for embryo transfer on Days 7-8. Immediately before ET, heifers were palpated for the presence of ovarian CL and CL papillae. Presence of CL, CL papillae, and CL lacunae were confirmed via transrectal B-mode ultrasound (Ibex EVO I, E. I. Medical Imaging). Transrectal colour Doppler ultrasound (Ibex EVO I, E. I. Medical Imaging) was used to evaluate blood flow to the CL. Invitro-produced (IVP) blastocysts (qualities I and II) were transferred to the uterine horn ipsilateral to the CL. All palpation, imaging, and embryo transfer was performed by a single technician. Pregnancy was determined via transrectal ultrasound 24-32 days post-embryo transfer (31-39 days of embryo age). Blood flow to the CL was split into three categories: high, median, and low. Presence or absence of two additional CL structures, CL papillae and lacunae, were recorded. Fisher's exact test was used to evaluate data. Statistical differences were considered significant at P&amp;lt;0.05. The overall pregnancy rate was determined to be 40%, 36 out of 90. Pregnancy rates were similar in high (33.9%, 20 of 59), median (59.09%, 13 of 22), and low (33.33%, 3 of 9) blood flow categories. Consistent with previous studies, absence of CL papillae and presence of CL lacunae at the time of ET did not affect pregnancy rate. Doppler blood flow, papillae, and lacunae were also evaluated together in each possible combination. Only one outlier was present; all other differences were not significant. The highest pregnancy rate (66.67%, 10 of 15) was observed in recipients with CLs with median Doppler blood flow, a palpable CL papillae, and no CL lacunae. A larger sample size is required to determine the accuracy of this measurement. In conclusion, recipients with high to low CL blood flow are suitable for IVP embryo transfer programs. Alone, colour Doppler imaging of the CL does not predict pregnancy rate.

Identification and clinico-gynaecological characterization of reproductive disorders in crossbred cows under field conditions

The present study was conducted on a total of 909 crossbred cows maintained in various private farms from Kamrup, Darrang and Lakhimpur districts of Assam. Breeding records provided by the farmers and characteristic genital changes detected on per rectum examination of 133 reported infertile cows were analyzed. The study revealed that the incidence of infertility due to different types of reproductive disorders was 14.63 per cent in cross bred cow under field conditions. Common clinically detectable reproductive disorders were repeat breeding with uterine infection, repeat breeding without uterine infection, true anoestrus and silent oestrus with the percentage frequencies of 36.09, 23.30, 20.30 and 17.29 respectively when estimated out of total number of infertile cows. Less frequent reproductive disorders were pyometra, ovarian cyst, infantile genitalia and ovario-bursal adhesion occurring in the frequencies of 36.09, 23.30, 20.30 and 17.29 per cent respectively. Irrespective of type the incidence of anoestrus and repeat breeding was 37.59 and 59.39 per cent respectively. Palpable corpus luteum was present in the ovary of silent oestrous cows but totally absent in cows affected with true anoestrus. Mucopurulent vaginal discharge was a distinct clinically detectable genital characteristic observed in 62.50 per cent repeat breeder cows with uterine infection. Of the infertile cows 53.38 per cent had body condition score between 2.5 and 3.5 and 46.61 per cent had the score less than 2.5. Out of repeat breeder cows with uterine infection 43.66 per cent had good body condition and 27.41 per cent poor body condition. Level of serum calcium, zinc, leptin, ghrelin and IGF-1 did not vary significantly between types of infertility. Level of phosphorus and iron was lower in cows affected with repeat breeding due to infection. Serum oestrogen level remained low in cows affected with silent oestrus and true anoestrus while level of progesterone was higher in cows affected with silent oestrus.

Effectiveness of Different Hormonal Treatment Protocols on Treatment of Postpartum Anoestrus in Cross-Bred Dairy Cows

Postpartum anoestrus (PPA) is one of the major factors causing prolonged calving interval &amp; that leading to decreased annual milk production. Hormonal treatments can be effective tools for treatment of PPA when nutritional &amp; environmental management are good. So, objective of this study was to know the comparative effectiveness of different hormonal protocols on treatment of PPA in cross-bred dairy cows. Used protocols were: single dose of GnRH, Ovsynch with TRIU-B and single dose of PGF2α injection which were used in three different groups, each containing ten animals. GnRH and Ovsynch with TRIU-B were used in postpartum true anoestrus cattle with inactive ovaries while PGF2α was used in postpartum silent oestrus condition having palpable corpus luteum and the condition were identifi ed through rectal palpation and history record. Cows treated with Ovsynch+TRIU-B showed higher oestrus expression rate within one week (80%) than cows treated with GnRH (10%) and PGF2α (50%). Ovulation rate was also comparatively higher in Ovsynch+TRIU-B group (100%) than GnRH (90%) and PGF2α (70%). But the pregnancy rate, checked after 45 days of first AI through rectal palpation, was comparatively higher in GnRH group (40%) than in Ovsynch+TRIU-B (30%) and PGF2α (20%). Among treated cows, pregnancy rate was found to be signifi cantly higher (P=0.02) in cows with ≥100 days duration of postpartum anoestrus (53.84%) than in cows with 100 days duration (0%). In conclusion, ovsynch with intravaginal progesterone (p4) releasing device was more effective at inducing oestrus and synchronizing ovulation than the single GnRH injection. However, single GnRH injection was as effective as ovsynch with intravaginal p4 releasing device for pregnancy outcomes in anoestrous crossbred dairy cows in a commercial dairy farm where formulated concentrate ration is daily fed.

Anti-Mullerian Hormone and Follicle Counts as Predictors of Superovulatory Response and Embryo Production in Beef Cattle

This study evaluated Anti-Mullerian hormone (AMH) and/or follicle counts as predictors of subsequent superovulatory response and embryo production in 79 beef cows. Before superovulation, 3 to 5 mm follicles presented on the ovaries of donor cows were counted, and blood was collected for measure of serum AMH. Across cows, serum AMH ranged from 0.013 to 0.898 ng/mL, with a mean of 0.293 ng/mL. The distribution of both AMH concentrations and follicle counts were divided into quartiles (AMH Q1 through Q4, with Q1 the lowest and Q4 the highest) for analysis. Donor cows in AMH Q4 had a greater (P < 0.001) number of 3 to 5 mm follicles at the start of superovulation than did donors in either Q1 or Q2. At embryo collection, cows in AMH Q3 and 4 had more (P < 0.001) palpable corpora lutea (CL) than cows in AMH Q1. The mean number of embryos recovered from donor cows in AMH Q4 was greater (P < 0.001) than those recovered from cows in either AMH Q1 or 2, but similar to that of AMH Q3. Analysis indicated that AMH was positively correlated (P < 0.001) with mean follicles (r = 0.458), CL (r = 0.452) and embryos recovered (r = 0.430). Donor cows with higher follicle counts (F Q3 and 4) at the start of superovulation had more (P < 0.001) palpable CL at embryo collection than donor cows in F Q1 or 2. More (P < 0.001) embryos were recovered from cows with the highest follicle counts (F Q4) as compared with cows having lower (F Q1 and 2) follicle counts. Results confirm that relative AMH concentration was positively correlated with number of small antral follicles in the ovaries of cows; both AMH measure and antral follicle counts might be used to predict subsequent superovulatory response in beef cows.

5 Live Offspring Produced from Reproductive Material Recovered During the Annual Cull of Bison from Yellowstone National Park

Bison from Yellowstone National Park (YNP) have a unique genetic history that makes them a conservation priority for the species. Unfortunately, there is a high prevalence of the zoonotic disease brucellosis in the herd. Part of the management strategy for controlling the disease and herd size in YNP is an annual culling process. This culling may result in loss of valuable genetic material and, until now, has been a missed opportunity to recover and preserve those genetics. The goal of this project was to demonstrate the feasibility of producing healthy offspring from reproductive material collected during the nonbreeding season from bison postmortem and to determine effects of donor parameters, including sexual maturity and pregnancy status on IVF outcomes. Oocytes were collected from ovaries harvested from bison within 2 h of slaughter during winters of 2014 to 2017 for use in in vitro embryo production (IVP). Frozen-thawed semen from 7 YNP bulls collected via electroejaculation in vivo or from epididymal flushes postmortem was used for IVF. Embryos were produced using standard cattle IVP procedures. On Days 7, 7.5, and 8 of in vitro culture, embryos were assessed for developmental stage and quality, and Grade 1 and 2 embryos were vitrified on Cryotops® (Kitazato, Tokyo, Japan) in a two-step equilibration process. Embryos were then stored until the breeding season when they were warmed, cultured for 6 h, evaluated for survival, and transferred to healthy bison recipients. Data were analysed using a Student’s t-test. Age did not affect the mean number of oocytes collected from a mature female (n = 254) v. juvenile (n = 27; 24 ± 16 v. 24 ± 13), embryo cleavage rates (54 ± 24% v. 45 ± 24%), or the rate of blastocyst development (8 ± 12% v. 7 ± 8%, respectively). Pregnancy status did have a significant effect on the number of oocytes collected per female (pregnant 22 ± 15%, n = 258; nonpregnant 26 ± 20%, n = 82; P = 0.04) and the percent of oocytes that cleaved (pregnant 56 ± 23%, n = 189; nonpregnant 47 ± 24, n = 61; P = 0.005). No difference was detected in the percent of embryos that reached the blastocyst stage of development (pregnant 8 ± 10%, nonpregnant 8 ± 15%). In August 2016, 12 embryos were transferred singly or in pairs to 10 recipient bison synchronized using a standard synch protocol. Ten days following removal of the CIDR, embryos were transferred to recipient bison with a palpable corpus luteum. In spring of 2017, a healthy female calf was born to a recipient who had received 2 Grade-2 blastocysts as graded at the end of the 6-h incubation period. The genetic dam of the calf was a pregnant adult bison cow. This calf demonstrates that live offspring can be generated from oocytes collected from bison postmortem in the nonbreeding season. Although cleavage rates were higher with oocytes from pregnant bison, viable embryos were generated from nonpregnant adults and juvenile females, warranting continued collection of their oocytes and banking of resulting embryos. To our knowledge, this is the first bison calf produced by in vitro production using postmortem reproductive material.

Effect of GnRH or Estradiol Benzoate on Reproductive Traits during a Heatsynch Protocol in Dairy Cows

Background: Milk production of cows is closely correlated with its reproductive efficiency. One of the several factors influencing the dairy efficiency is the decline in fertility of the cows/heifers. Use of hormonal protocol are often used to improve the fertility of the recipients. Several programs are available to inseminate dairy cows, with variation in the use of different types of hormones, time of use and range of duration period, as well as the time of AI. The objective of this study was to evaluate the efficiency of follicular wave emergence induction of estradiol benzoate in comparison to GnRH in a Heatsynch protocol and its influence on reproductive parameters.Materials, Methods &amp; Results: In this study were used primiparous and multiparous Holstein cows (n = 325). Animals were divided randomly into two different treatments according with the hormone used to induce follicular wave emergence (D0). The GnRH group (n = 167), was given 25 mg, while in the EB group (n = 158) was used 2 mg. At same day, were inserted, a controlled internal drug-releasing containing 1.9 g of progesterone CIDR®. On day 7, CIDR was removed and cows were given 25 mg luteinizing hormone followed by an injection of 1 mg Inducer Ovulation at day 8. Cows were then observed for signs of estrus for at least two hours (twice daily at 7 am and 6 pm) for three days following CIDR® removal. Cows detected in estrus were inseminated 12 h later the estrus detection. Pregnancy status was determined by ultrasonography at 30 ± 5 d and 60 ± 5 d after AI. A single technician was responsible for the estrus detection and inseminations. A single technician was responsible for the estrus detection and inseminations. Ultrassonographic examinations with ultrasound, transrectal linear probe of 5 MHz, were performed for the diagnosis of pregnancy at 30 and 60 days after artificial insemination. Binomially distributed data, such as, estrous response, conception rate, pregnancy rate and pregnancy losses, were analyzed by Chi-square test. Pregnancy rates at 30-35 days and 60-65 days as well as pregnancy losses were considered in the model for the effect of parity (primiparous vs. multiparous) and the group of estrus behavior during the Heatsynch protocol. Conception rate was similar between GnRH (36.1%, 57/158) and EB (37.7%, 63/167) treatments. Cows showing estrus signs after CIDR removed were 71.9% in GnRH and 62% in EB group (P = 0.05). Embryonic losses were different (P = 0.03) according to groups of cows showing estrus signals or not.Discussion: Our results demonstrate that conception rates are not affected by using either EB or GnRH at the beginning of the Heatsynch protocol. Therefore, the EB can be an alternative choice to GnRH at Heatsynch beginning. Similar results were found by autor in dairy cows, once it was also observed that there is no difference in conception rate in two TAI protocols using either GnRH or EB. In Heatsynch protocols the increase in estrus detection are a key factor to increase fertility in lactating dairy cows. It can be explained once animals in regular estrous cycle have a palpable corpus luteum and circulating progesterone concentration higher than 1 ng/mL which leads than to enhance pregnancy ratios. The use of BE at the time of CIDR insertion in TAI protocols for dairy cattle proved to be an alternative to GnRH considering conception rates. Therefore, the use of BE at the time of CIDR insertion in a Heatsynch protocol in dairy cattle proved to be an alternative to GnRH which can be an important tool to increase the TAI utilization by farmers. Furthermore, in Heatsynch protocol cows showing estrus have pregnancy rates improved and lower pregnancy losses.

233 ANTI-MÜLLERIAN HORMONE AS A PREDICTIVE ENDOCRINE MARKER FOR SUPEROVULATORY RESPONSE AND EMBRYO PRODUCTION IN BEEF CATTLE

A study investigated the use of anti-Müllerian hormone (AMH) and/or follicle counts as a predictor of subsequent superovulatory response and embryo production in 79 beef cows. Before initiation of superovulation, ultrasonography was used to scan the ovaries of each donor cow to record the number of 3 to 5 mm follicles present, and a blood sample was collected for measure of serum AMH. At the time of embryo collection, the ovaries of donor cows were palpated to estimate the number of corpora lutea (CL) present on each ovary. Recovered embryos were evaluated for stage of development and morphological quality. Across cows, serum AMH ranged from 0.013 to 0.898 ng mL–1, with a mean of 0.293 ng mL–1. The distribution of AMH concentrations was divided into quartiles (AMH Q1 through Q4, with Q1 the lowest and Q4 the highest, ng mL–1) for analysis (ANOVA). Number of donors/collections in AMH Q1 through Q4 was 26, 22, 24, and 24, respectively. Donor cows in AMH Q4 had a greater (P &lt; 0.001) number of 3- to 5-mm follicles at the start of superovulation than did donors in either Q1 or Q2. At embryo collection, cows in AMH Q3 and 4 had more (P &lt; 0.001) palpable CL than cows in AMH Q1. The mean number of embryos recovered from donor cows in AMH Q4 (20.1 ± 1.8) was greater (P &lt; 0.001) than those recovered from cows in either AMH Q1 (9.8 ± 1.8) or 2 (9.4 ± 1.9), but similar to that of AMH Q3 (15.5 ± 1.8). Percentages of recovered embryos classified as transferrable, degenerate, or unfertilized were similar (P ≥ 0.275) across AMH quartiles. Analysis indicated that AMH was positively correlated (P &lt; 0.001) with mean follicles (r = 0.458), CL (r = 0.452), and embryos recovered (r = 0.430). To determine if follicle counts at the start of superovulation are predictive of superovulatory response, the distribution of follicle counts was divided into quartiles (F Q1 through Q4, with Q1 the lowest and Q4 the highest) for analysis. Donor cows with higher follicle counts (F Q3 and 4) at the start of superovulation had more (P &lt; 0.001) palpable CL at embryo collection than donor cows in F Q1 or 2. More (P &lt; 0.001) embryos (20.0 ± 2.2 v. 9.6 ± 1.8 and 11.6 ± 1.6) were recovered from cows with the highest follicle counts (F Q4) compared with cows having lower (F Q1 and 2) follicle counts, respectively. The percentage of transferable embryos and unfertilized ova were similar (P ≥ 0.688) across follicle count quartiles. As was noted for AMH, mean number of follicles at the start of superovulation was positively correlated (P &lt; 0.001) with mean CL (r = 0.556) and mean embryos (r = 0.423) but not percentages of viable or degenerate embryos or unfertilized oocytes (P ≥ 0.153). Results confirm that relative AMH concentration was positively correlated with number of small antral follicles in the ovaries of cows and might be used to either predict superovulatory response or possibly adjust superovulatory regimen to improve superovulatory response. Further study is needed to determine the effectiveness of using either AMH concentration or follicle counts to adjust superovulatory regimens.

Influence of controlled breeding techniques on fertility and plasma progesterone, protein and cholesterol profile in true anestrus and repeat breeding buffaloes

Aim: The aim was to evaluate the estrus response, conception rate and plasma profile of progesterone, protein and cholesterol following use of different hormonal protocols in anestrus and repeat breeding buffaloes. Materials and Methods: This study was carried out on 20 true anestrus, 20 repeat breeders, and 10 normal cyclic buffaloes. Ten anestrus buffaloes each were treated with standard controlled internal drug releasing (CIDR) i/vg device and Ovsynch (GPG) protocols with fix timed artificial insemination (FTAI), and blood samples were obtained on day 0, 7, 9/10 (AI) of treatment and day 21 post-AI. Ten repeat breeding buffaloes with mature mid-cycle palpable corpus luteum (CL) were treated with i/m injection of 25 mg prostaglandin F2α (PGF 2 α) with FTAI twice at 72 and 96 h later, whereas other ten repeat breeding buffaloes in standing estrus were inseminated with simultaneous i/m injection of buserelin acetate-gonadotropinreleasing hormone (GnRH) 20 μg. 10 buffaloes exhibiting first estrus within 90 days postpartum and inseminated without any treatment served as normal cyclic control. Blood samples were obtained on day of PG injection, day of AI and day 21 post-AI for estimation of plasma progesterone, protein, and cholesterol. Results: CIDR and Ovsynch protocols resulted in 100 and 80% induction of estrus with conception rates of 40 and 30% at induced estrus, respectively, in anestrus buffaloes. Mid-cycle PGF 2 α treatment resulted in 90% estrus induction and 40% conception rate at induced estrus, while Buserelin acetate-GnRH 20 μg injection at AI resulted in 30% conception rate in repeat breeders. In normal cyclic control group also, the first service conception rate was 30%. The mean plasma progesterone concentrations on day 0, 7, 9/10 (AI) of treatment and on day 21 post-AI were found to be significantly (p<0.05) different in both CIDR and Ovsynch protocols, being higher on day 7 (day of PG injection) and on day 21 post-AI than on day 0 and 9/10 (FTAI), which were near basal levels. The mean plasma progesterone level was significantly (p<0.01) higher on the day of initiation of mid-cycle PGF 2 α treatment (3.81±0.67 ng/ml) in a repeat breeding buffaloes suggesting luteal phase. The mean plasma P 4 levels on day 21 post-AI were significantly (p<0.01) higher than on the day of estrus in both repeat breeders and in normal cyclic controls. The plasma P 4 value on day 21 post-AI was significantly (p<0.01) higher in conceived than non-conceived buffaloes in all five groups. The mean plasma total cholesterol and total protein concentrations in anestrus and repeat breeding buffaloes under different treatments did not vary significantly between sampling days. However the cholesterol content was significantly (p<0.05) lower (79.96±2.17 vs. 92.27±6.04 mg/dl) and protein higher (8.14±0.73 vs. 7.69±0.59 g/dl) in conceived than non-conceived animals. In both anestrus and repeat breeding buffaloes, the values of cholesterol and protein were significantly lower than in normal cyclic control group (138.04±11.98 mg/dl and 7.82±0.11 g/dl, respectively). Conclusion: The results showed that CIDR was better than Ovsynch protocol in inducing fertile estrus in anestrus buffaloes, while mid-cycle PG treatment was superior over AI + GnRH in repeat breeders, and all four treatment protocols significantly influenced plasma P4 profile, but not the protein or cholesterol.

311 SUPEROVULATION OF NORTH AMERICAN BISON WITH TWO INJECTIONS OF FOLLICLE-STIMULATING HORMONE

Few studies have examined superovulation of North American Bison. In cattle, ovarian superstimulation is usually achieved with 6 to 8 injections of FSH at half-day intervals. However, handling bison repeatedly stresses the animals, which could adversely affect their reproductive potential, as well as pose a risk of injury to the bison and handlers. To limit the number of times the bison were handled, we tested a two-injection superovulation scheme using sodium hyaluronate (MAP-5, Bioniche Animal Health Inc., Belleville, Ontario, Canada) in the FSH diluent, which serves to slow the absorption of FSH when given IM. We hypothesised that the two-injection superstimulation protocol would result in recovery of more embryos on average than a single-embryo recovery protocol. Although a traditional superovulation scheme with 6 to 8 injections of FSH would have been a better comparison, our goal was to handle the bison minimally. Eight female bison ranging in age from 5 to 11 years were used as embryo donors. For superovulation, females with a corpus luteum (CL) were given 25 mg of prostaglandin F2α (PGF, Lutalyse, Pfizer Animal Health, New York, NY, USA) IM followed by 266 mg of FSH (Folltropin V in MAP-5 diluent, i.m., Bioniche Animal Health Inc.) 12 days later (or 9 days after presumed oestrus 3 days post-PGF). Forty-eight hours after the first FSH injection, 134 mg of FSH IM and 25 mg of PGF IM were given. Two days later females were put in a pen with a bison bull for natural breeding. Seven days after assumed oestrus, embryos were recovered nonsurgically. Although the situation is not clear in bison, there is evidence in cattle that superovulated cycles influence embryo collections in subsequent cycles. Consequently, females were randomly assigned to a superovulation or single-embryo recovery treatment for each cycle; however, consecutive superovulation protocols were never conducted without a short oestrous cycle in between. Superovulated females were given PGF at embryo recovery after superovulation, followed by PGF 12 to 14 days later, and bred off the assumed oestrus of the second PGF injection. Embryos were collected from females 4 times (2 superovulation and 2 single embryo cycles, except one bison that was superovulated once). Data were analysed using a one-tailed t-test. Superovulation resulted in greater mean numbers of palpable CL (3.7; P &lt; 0.001), embryos collected (1.8; P &lt; 0.05), and transferable quality embryos (0.8; P &lt; 0.05) compared with the single-embryo recovery protocol (mean palpable CL, 1.0; embryos collected, 0.5; transferable embryos, 0.2). Notably, the bison breeding season is July to September and occasionally animals breed in October; this experiment was conducted in October and November. Thirteen transferable embryos were nonsurgically transferred to recipients; 6 pregnancies were established, but 5 were resorbing by 2 months of gestation; 1 healthy calf was carried to term. Two injections of FSH with a long-acting diluent can be used to increase the number of embryos recovered from bison compared with a single-embryo recovery scheme.

43 COSTS RELATED TO ESTRUS SYNCHRONIZATION AND AI PROTOCOLS IN CROSSBRED HOLSTEIN-ZEBU DAIRY COWS

The objective of this paper was to compare 3 bovine AI protocols, evaluating the cost of each treatment and the pregnancy rates obtained, to demonstrate the importance of routine utilization of estrus synchronization techniques. The experiment was conducted in the Farm School of Cachoeiras de Macacu of the Federal Fluminense University, in the state of Rio de Janeiro, during the period from 2006 to 2007 (August to June). One hundred sixteen crossbred female Holstein-Zebu dairy cows in cycle, with body condition between 2.5 to 4 (on a scale from 1 to 5), were selected and divided randomly in 3 groups (G). Animals in G1 [n = 60; 21 heifers (H), 13 primiparous (P), and 26 multiparous (M)], with palpable corpus luteum, received 25 mg of prostaglandin F2 intravulvar, and AI was done 12 h after estrus detection. For animals in G2 (n = 30; 11 H, 6 P, and 13 M) an intravaginal progesterone device (CIDR®, Pfizer Animal Health, New York, NY, USA) was inserted and 2 mg of estradiol benzoate (EB) was administered i.m. on Day 0. On Day 8 the device was removed and 25 mg of prostaglandin was administered i.m. on Day 9, 2 mg of EB was administered, and 30 h later, timed AI of all animals was performed. For G3 (n = 26; 9 H, 5 P, and 12 M), the protocol used was similar to that for G2, except that on Day 8 400 IU of eCG was administered (in addition to the removal of the device) and on Day 9 the dose of EB was reduced to 1 mg and timed AI was done 27 h later. AI was done with semen from 2 sires with proven fertility, by the same technician. The pregnancy diagnoses were done on Day 45 by palpation per rectum and transrectal ultrasonography. The cost-benefit relation was calculated according to the cost of pharmaceuticals multiplied by the number of animals in each experimental group divided by the number of pregnancies obtained in each one, without considering the manual labor costs in all the groups and considering 3 uses of the CIDR. The statistical analyses were done using chi-square test. The pregnancy rate obtained in G2 (60%, 18/30) was superior to that in G1 (30%, 18/60) (P &lt; 0.05), demonstrating the efficiency of the treatment that utilized progesterone. G3 (53.85%, 14/26) was similar (P &gt; 0.05) to G2, despite the fact that the eCG was added in this group. In reference to cost-benefit, considering only the pharmaceuticals costs and the value for pregnancy obtained in each group, the costs found, respectively, were G1 U$7.02 and U$23.66; G2 U$28.57 and U$47.59; and G3 U$47.96 and U$89.05. The lowest cost in the estrus synchronization was based on the use of prostaglandins alone; the use of progesterone increased the synchronization cost, but also increased the pregnancy rate and eliminated the need for estrus observation. The addition of eCG in cows that are cycling does not necessarily demand a increase in pregnancy rate. Failure in estrus detection, using prostaglandin alone, increased the interval from calving, causing a daily loss in milk production, which justified the investment in protocols of estrus and ovulation synchronization, contributing to increase the reproductive efficiency of a dairy cow herd.

Effect of addition of hyaluronan to embryo culture medium on survival of bovine embryos in vitro following vitrification and establishment of pregnancy after transfer to recipients

Two experiments were conducted to determine whether addition of hyaluronan to culture medium could improve survival of bovine embryos after vitrification or following embryo transfer. In Experiment 1, embryos were produced in vitro and cultured for 7 days in modified synthetic oviductal fluid (SOF) containing one of four concentrations of hyaluronan (0, 0.1, 0.5, or 1 mg/mL), with or without 4 mg/mL of bovine serum albumin (BSA). On Day 7 after insemination, blastocysts and expanded blastocysts were vitrified using open-pulled straws. At a concentration of 1 mg/mL, hyaluronan increased ( P < 0.05) the percentage of oocytes that were blastocysts and re-expansion rate at 24 h after warming. At 0.5 mg/mL, hyaluronan tended ( P < 0.10) to increase re-expansion rate at 48 h after warming and increased ( P < 0.05) embryo hatching rate at 24 and 72 h. Treatment with BSA caused a slight reduction in cleavage rate ( P < 0.05), but only for cultures containing hyaluronan (BSA × hyaluronan, P = 0.10), an increase in the percentage of oocytes that became blastocysts ( P < 0.001), and a reduction in re-expansion rates ( P < 0.001) and hatching rates ( P < 0.05 or P < 0.01) at all times examined. In Experiment 2, embryos were produced in vitro and cultured in modified SOF containing 4 mg/mL BSA, with or without 1 mg/mL hyaluronan. At 159–162 h after insemination, grade 1 morula, blastocysts and expanded blastocysts were harvested for embryo transfer. Harvested embryos were transferred individually to lactating Holstein recipients with a palpable corpus luteum on Day 7 after presumptive ovulation. There was an interaction ( P < 0.05) between hyaluronan and embryo stage on pregnancy rate. Recipients that received morula and blastocyst stage embryos treated with hyaluronan had a higher pregnancy rate than recipients that received control embryos of the same stage. There was no effect of hyaluronan on pregnancy rates of recipients that received expanded blastocysts. In conclusion, addition of hyaluronan to embryo culture enhanced blastocyst yield, improved survival following vitrification, and enhanced the post-transfer survival of fresh morula and blastocyst stage embryos.

Hormonal profiles associated with treatment of cystic ovarian disease with GnRH and PGF2α with and without CIDR in dairy cows

This study aimed to investigate ovarian and endocrine responses associated with treatment of cystic ovaries with GnRH, PGF2α, and +/-Progesterone-CIDR placement; and to determine pregnancy establishment following synchronization and timed artificial insemination in cows suffering from cystic ovaries. Ovarian cysts were typed rectally and ultrasonographically into follicular, luteinized and persistent types. Two schedules of treatment were designed, in Schedule-A, 15 cows with follicular (n=7), luteinized (n=3) and persistent (n=5) cysts were used. The cows received 2 injections of 100 ug GnRH (9 days interval) with 25 mg PGF2α administered 7 days after 1 st GnRH dose. In Schedule-B, 14 cows with follicular (n=6), luteinized (n=3) and persistent (n=5) cysts were used. The cows administered GnRH and CIDR concurrently, then 25 mg PGF2α 7 days later. The CIDR was removed 2 days after PGF2α. Cows were inseminated 16 hours after 2 nd GnRH dose. Blood samples were collected to assay progesterone and estradiol levels. In schedule-A, 57.1%, 33.3% and 40% of follicular, luteinized and persistent cysts were ovulated after 1 st GnRH injection, while a total of 100%, 66.7% and 100% were ovulated after 2 nd GnRH dose. The cows with follicular cysts appeared the highest rate of pregnancy (57.1%) followed by the luteinized and persistent cysts (33.3% and 40%). Of a total 15 cystic-treated cows, 7 were confirmed pregnant (46.7%). The concentration of progesterone was significantly (P<0.05) higher at 1 st GnRH injection, then decreased after PGF2α injection and then increased again at 7 day after insemination. In schedule-B, after 1 st GnRH dose, 66.7% of follicular, 0% of luteinized cysts and 80% of persistent were ovulated; with a 57.1% total ovulation rate. Following CIDR removal, 85.7% (12/14) cows ovulated the recruited follicles and developed a corpus luteum. Of a total 14 cystic-treated cows, 8 were confirmed pregnant (57.1%). Plasma estradiol declined following intra vaginal placement of CIDR, but markedly increased one day after removal. Progesterone decreased after CIDR removal, but increased again at presence of palpable corpus luteum. The developed corpus luteum and pregnancy rate were higher in schedule-B (+CIDR) than in schedule-A (-CIDR). In conclusion, treatment of cows with cystic ovaries with GnRH followed by PGF2α 7 days later resulted in recruitment of new healthy follicles, synchronization of ovulation and a marked pregnancy rate (with than without CIDR).

Calves Born from Anestrus Yaks (Poephagus grunniens L.) Subjected to Ovsynch and Superovulation Treatment

An attempt was made to induce estrus and ovulation in eight anestrus yaks by use of the Ovsynch protocol. Six out of eight yaks were successfully induced into estrus, and ovulation occurred in all the responding yaks 1-2 days after the second GnRH administration. Out of the six animals that responded to the treatment, two mated naturally with yak bulls, and calves were obtained from them. The other four animals were further administered a superovulatory regimen of Folltropin (FSH-P). Following Folltropin and Ilerin (a PGF(2alpha) analog) treatment, the animals were subjected to natural insemination. Only one animal in which natural mating occurred was flushed non-surgically for embryo recovery 7 days post-insemination. Thereafter, all the donor animals were administered with Ilerin. After 48-72 h, they came into heat and mated naturally with yak bulls, and calves were obtained from them after expiration of the normal gestation period. Following superovulation, the average numbers of palpable corpora lutea in the right and left ovaries were 2.25+/-0.6 and 1.75 +/-0.3, respectively. Three embryos were recovered by non-surgical flushing from a single animal. One embryo was transferred to a recipient yak, who produced one female calf after 258 days. This is the first report of production of a yak calf through embryo transfer-technology.

Reproductive performance in the subsequent lactation of dairy cows previously treated for failure to be detected in oestrus

AIM: To describe the effect of treatment of cows not detected in oestrus (NDO) with progesterone (P4) and oestradiol benzoate (ODB) in one lactation, on the reproductive and productive performance in the subsequent lactation. METHODS: Cows (n=770 from nine herds) which were NDO 10 days before the planned start of the seasonal breeding programme (PSM) were blocked by herd, age (2 or >2 years old) and ovarian status (i.e. with or without a palpable corpus luteum; CL), and treated either with an intravaginal progesteronereleasing device and ODB and resynchrony, or were left as untreated controls. In the following lactation, data were collected on the occurrence of endometritis, NDO, breeding dates, pregnancy test results and milk production. The effect of treatment and ovarian status in the previous lactation on the incidence of disease, and the proportions submitted for service, conceived and pregnant in the subsequent lactation were analysed. Calving dates and intervals from start of breeding to first insemination and conception were analysed using Kaplan Meier survival analysis. Additional multivariate analyses were undertaken to include known confounders such as age, breed, herd, calving date and milk production, as well as the cyclic status (i.e. previously cycling, NDO, or ‘late-calving’) in the previous lactation, to examine the potential ‘carryover’ effects of previous status on the reproductive performance in the subsequent lactation. RESULTS: Treatment of NDO cows resulted in an earlier calving date (Julian calving date 214, 95% CI=207–221, vs 224, 95% CI=220–228; p=0.005), more female calves reared (31.4% vs 23.3%; p=0.01), and reduced risk of being NDO (33.9% vs 46.1%; p=0.002) in the subsequent lactation compared with controls. There were no differences in the incidence of peripartum disease, or the proportion of cows submitted, conceived or pregnant between the Treatment and Control groups in the subsequent lactation (p>0.2). Previously CL-positive (+ve) NDO cows were less likely to produce a female calf that was reared (19.0% vs 29.9%; p=0.005), more likely to be treated as NDO (26.4% vs 18.6%; p=0.02), less likely to conceive by the end of the subsequent lactation's breeding programme (82.2% vs 87.8%; p=0.09), and took longer to conceive (41 vs 33 days from the PSM to conception) than cows that were NDO-CL-negative (-ve) in the previous lactation. There was no interaction between ovarian status and treatment for any of the outcome variables (p>0.05). Cows that were NDO or late-calving in the previous lactation were more likely to be NDO (p<0.005), treated as NDO (p<0.005), and have longer PSM Animal Health Centre, PO Box 21, Morrinsville, New Zealand. Author for correspondence. Email: smcdoug@ahc.co.nz to conception intervals (p<0.005) in the subsequent lactation than cycling herdmates, despite inclusion of factors known to confound reproductive performance in analyses. CONCLUSIONS: Treatment of NDO cows resulted in more female calves reared, earlier calving and a reduced risk of being NDO in the subsequent lactation. Status (i.e. NDO-CL+ve or NDO-CL-ve) affected some reproductive measures in the subsequent lactation, and the effect of treatment in the subsequent lactation differed between the two groups for some measures. Reproductive performance in the subsequent lactation was not improved despite the earlier calving date and longer calving to PSM intervals. Additionally, the previous season's status was still important in the subsequent season's reproductive performance, despite adjusting for known confounders of fertility. CLINICAL RELEVANCE: The economic benefits from treatment of NDO cows are likely limited to effects of an earlier calving date and potential for longer lactation and increased milk yield, increased number of calves derived from artificial insemination and from a reduced number of NDO cows in the subsequent lactation. Effects of improved reproductive performance in the subsequent lactation were not demonstrated and hence should not be included in any economic analysis of therapy of NDO cows.

Effect of Different Prostaglandin Treatment Protocols on Luteolysis and Ovulation in Dairy Cows

During the past 25 years, several methods were developed to synchronize estrus in dairy cattle. Synchronization with prostaglandin F2α (PGF2α) is successful when cows are bred at a detected estrus because estrus detection rates and artificial insemination (Al) are more efficient than daily detection of estrus (Stevenson and Pursley, 1994). The success of estrous induction with PGF2α depends on the presence of a functional corpus luteum (CL). In case of a palpable CL, Archbald et al (1994) found that the percentage of milking cows observed in estrus within seven days after treatment (25 mg of PGF2α) was 55% (61/111). This management tool still does not control the time of AI, because estrus detection continues to be necessary, which is evidenced by the lower pregnancy rate after timed AI compared with AI after detected estrus. This might be partially explained by the variation in time of ovulation over periods of five days with respect to time of AI (Stevenson et al, 1987). Various attempts have been made to overcome this variability in response to PGF2α treatments. The administration of other hormones in conjunction with PGF2α, such as progesterone, estradiol benzoate, human chorionic gonadotropin (hCG) and gonadotropin releasing hormone (GnRH) (Deletang 1975; De Rensis and Peter 1999; Pursley et al, 1996), have been attempted. There was a better degree of synchronization but the pregnancy rate was similar to that of untreated cows. Similarly, two prostaglandin injections at an eight-hour interval was more effective on the incidence of luteolysis than a single injection (Archbald et al, 1993). There are several studies dealing with the effect of prostaglandin on the area changes of corpus luteum and follicles in heifers (Kastellic and Ginther 1991), on corpus luteum size and plasma progesterone concentrations in cattle (Assey et al, 1993), and the effect of different doses of PGF2α on fertility (Lagar 1977), however the effect of different doses of PGF2α on the area changes of the corpus luteum, the largest follicle and the progesterone (P4) concentration has not been examined. The objective of our study was to compare the effect of different doses of PGF2α (first trial-0 mg, 25 mg, 35 mg; second trial-2 x PGF2α treatment at an eight-hour interval) on the corpus luteum, the largest follicle and the progesterone concentration in dairy cattle. The time to ovulation after AI in PGF2α-treated and untreated cows (third trial) was also determined.

The sensitivity and specificity of single manual examination tests for detection of corpora lutea in the ovaries of non-cycling cows

To evaluate the sensitivity and specificity of single manual examination of ovarian surface structures and of ovarian size as tests for the detection of corpora lutea in non-cycling cows Non cycling cows were dichotomously classified for the presence of palpable corpora lutea and for ovarian size. The performance characteristics of the palpable corpus luteum test and the ovarian size test were estimated using Bayesian analysis. Previously published information on prevalence and test performance was combined with observed data using the Gibbs Sampler to derive posterior distributions for prevalence and test parameters. Prior distributions for the prevalence of corpora lutea in cows not detected on heat before mating within seasonal herds were centred on 25%, and for the corpus luteum test sensitivity were centred on 70%. No prior assumptions for any other test parameter were made. From a total of 650 cows, 144 were found to have at least one corpus luteum, and 156 were found to have two small ovaries. The posterior estimate obtained for prevalence was 0.30 (95% Cl 0.22 to 0.43), for corpus luteum test sensitivity was 0.71 (95% Cl 0.49 to 0.93), and for corpus luteum test specificity was 0.98 (95% Cl 0.49 to 1.00). For the ovarian size test, the posterior estimate for size test sensitivity was 0.98 (95% Cl 0.95 to 1.00) and for size test specificity was 0.34 (95% CI 0.28 to 0.43). Sensitivity analysis indicated that corpus luteum test sensitivity ranged from 0.60 to 0.70 in most herds. Errors associated with pre-mating heat detection are likely to result in between 15 to 30% of cycling cows not detected in oestrous before mating, resulting in the inclusion of cycling cows within the population of non-cycling cows. This mixed population of cows is then subjected to manual examination by veterinarians in order to assign cows to treatment groups. The corpus luteum test has modest sensitivity and high specificity and the size test has high sensitivity and low specificity. Therefore the use of a single examination of the ovaries of cows not detected on heat before the mating period, in order to classify them as anovulatory anoestrous cows with or without a corpus luteum, is not sufficient to accurately classify them and thus to recommend treatment.

189 PREGNANCY RATES IN HEAT-STRESSED DAIRY CATTLE RECEIVING ONE OR TWO IN VITRO-PRODUCED EMBRYOS IN A TIMED EMBRYO TRANSFER PROGRAM

The objective was to determine whether transfer of two embryos would increase pregnancy rates in heat-stressed dairy recipients receiving an in vitro-produced embryo transferred into the uterine horn ipsilateral to the corpus luteum (CL). Such a treatment would increase the likelihood that the cow receives at least one embryo competent for sustained development. In addition, transfer of two embryos into the ipsilateral uterine horn is likely to increase the amounts of interferon-tau and other embryonic-signaling molecules in the uterus. A total of 32 virgin crossbreed heifers and 26 lactating crossbreed cows were used as timed embryo transfer recipients after being subjected to an ovulation synchronization protocol as follows: GnRH (100 �g) and insertion of previously used progesterone-containing CIDR on Day -10, prostaglandin F2� and CIDR removal on Day -3, and GnRH (100 �g) on Day 0 (day of anticipated ovulation). All recipients had a palpable CL on Day 6 and were randomly selected to receive one (n = 31 recipients) or two (n = 27) embryos on Day 7. At Day 64, the pregnancy rate tended to be higher (P = 0.07) for cows than for heifers. While not significant, heifers that received two embryos tended to have lower pregnancy rates than those that received a single embryo (20% vs. 41%); there was no difference in pregnancy rate in cows (50% for two embryos vs. 57% for one embryo). Pregnancy losses between Day 64 and Day 127 occurred in one group only cows receiving two embryos. In that group, pregnancy rate was 50% at Day 64 but 17% at Day 127. Overall, there was no difference in pregnancy rates at day 127 between cows and heifers, but recipients that received two embryos (17% for cows and 20% for heifers) had lower pregnancy rates (P &lt; 0.03) than recipients that received one embryo (57% for cows and 41% for heifers). Only one animal, a cow, had twin fetuses at day 127. In conclusion, unilateral transfer of two embryos failed to improve pregnancy rates of dairy cattle exposed to heat stress. The fact that fetal loss occurred sooner for heifers than for cows points out the importance of uterine capacity as a limiting factor for maintenance of fetal development of two conceptuses. The suitability of timed embryo transfer was evident from the high pregnancy rates achieved with crossbreed females that received a single embryo. This work was supported by BARD Grant No. US-1551-14, USDA TSTAR Grant No. 2004-14135-14715, Grant No. 2001-12101-11318 from the USDA-IFAFS Program, and CAPES Grant No. 134202-1).