Circulating progesterone at insemination and accessory spermatozoa are associated with fertilization and embryo quality five or six days post insemination in dairy cattle.

This study aimed to evaluate animal and ovarian parameters that affected fertilization and embryo development up to the morula stage. The data were combined from four studies in which cows were inseminated between 46 and 60 days in milk, following a synchronization protocol, and flushed for embryo collection five or six days later. Parity and body condition score on the day of insemination were recorded. Cyclicity, ovarian structures, and circulating hormone concentrations before and on the day of insemination were also assessed. The recovered structures were graded on a 5-point scale (excellent-good quality, fair quality, poor quality, degenerated, and not fertilized). For recovered embryos, the total number of blastomeres, the number of nonviable blastomeres, and the number of accessory spermatozoa were assessed by epifluorescence microscopy. The risk factors for fertilization and embryo quality were identified using cumulative link mixed models. A total of 418 structures from 389 lactating Holstein cows (34% primiparous and 66% multiparous) were recovered. Thirty-five percent of the recovered structures were excellent-good quality embryos, 21% were fair quality embryos, 11% were poor quality embryos, 16% were degenerated embryos, and 17% were unfertilized oocytes. Structures from primiparous cows, from those with greater progesterone concentration at insemination, and from cows with seven or less accessory spermatozoa were less likely to be fertilized or of better quality than structures from multiparous cows, from those with lower progesterone concentration in plasma, and from those with more than seven accessory spermatozoa, respectively. Embryos with more blastomeres or without nonviable blastomeres were more likely to be of better quality than embryos with fewer blastomeres or with nonviable blastomeres. The results of this analysis highlight the importance of low circulating concentrations of progesterone near artificial insemination and potential positive association with number of accessory spermatozoa reaching the embryo and improvement of embryo development up to the morula stage.

Impact of gonadotropin-releasing hormone administration at the time of artificial insemination on conception risk and its association with estrous expression.

Cows with reduced estrous expression have compromised fertility. The aim of this study was to determine whether the administration of GnRH at the time of artificial insemination (AI) would affect ovulation rates and the fertility of animals expressing estrous behavior of lesser intensity. Cows were enrolled at the time of estrus from 3 farms (n = 2,607 estrus events; farm A: 1,507, farm B: 429, farm C: 671) and randomly assigned to receive GnRH at AI or not (control). The intensity of estrous expression, monitored through leg-mounted activity monitors, was determined using the maximum activity during estrus; estrous expression was categorized as greater or lower relative to the farm median. On farm A, cows were assessed at alert, and 24 h, 48 h, and 7 d post-alert for ovulation using ultrasonography. Pregnancy per AI was confirmed at 35 ± 7 d post-estrus for cows that were inseminated. Differences between treatments were tested using the GLIMMIX procedure of SAS. Treatment with GnRH at the time of AI increased pregnancy per AI (41.3 ± 1.6 vs. 35.7 ± 1.7%). An interaction between treatment and estrous expression on pregnancy per AI was found. Control cows with greater estrous expression had greater pregnancy per AI than those with lesser expression, whereas GnRH administration increased pregnancy per AI for cows with lesser estrous expression but not those with greater expression (GnRH, greater intensity: 43.5 ± 2.1; GnRH, lesser intensity: 37.8 ± 2.2; control, greater intensity: 42.6 ± 2.2; control, lesser intensity: 31.0 ± 2.2%). A higher proportion of cows with greater estrous expression that were administered GnRH at AI were found to ovulate by 48 h and 7 d post-estrus; however, ovulation of cows with lesser estrous expression was unaffected by GnRH administration. In conclusion, fertility of cows with reduced estrous expression may be increased using GnRH at the time of AI; however, increased ovulation rates do not seem to be the direct mechanism behind this relationship.

Plasma concentrations of progesterone in the preceding estrous cycle are associated with the intensity of estrus and fertility of Holstein cows.

The aim of this study was to determine the association between concentrations of progesterone (P4) during previous the estrous cycle with the intensity of spontaneous or estrogen-induced estrous expression and pregnancy per artificial insemination (P/AI). A total of 1,953 AI events from lactating Holstein cows were used, consisting of 1,289 timed AI events from experiment 1 (Exp. 1) and 664 AI events from experiment 2 (Exp. 2). In Exp. 1, cows were bred after a timed AI protocol based on estradiol and P4. In Exp. 2 animals were bred upon spontaneous estrus detection. In both experiments cows were continuously monitored by an automated activity monitor (AAM), in Exp.1 a relative increase of activity was calculated (i.e., percentage of increase activity at estrus compared to cow's baseline activity) and in Exp.2, activity data from each cow were computed into an index value that ranged from 0 to 100. In Exp.2 duration (hours) of estrus were calculated and defined as the total time above the threshold (35 index). The intensity of estrous expression was determined for each event and classified as either high or low intensity using the median of each experiment. Blood samples were collected for P4 analysis in Exp. 1 at -4 d, 0 d, and 7 d relative to timed AI, and in Exp. 2 immediately following estrus (0 d), 7 d, 14 d, and 21 d post-AI. Concentration of P4 was classified as greater or lower according to the median value in each experiment. Cows with lower concentrations of P4 at AI had greater estrous expression in Exp. 1 (363.6 ± 5.2 vs. 275.9 ± 8.0% relative increase) and Exp. 2 (76.7 ± 1.9 vs. 67.4 ± 4.7 index; and 12.5 ± 0.5 vs. 9.3 ± 1.8 hours). Cows with a greater intensity of estrous expression at timed AI had greater concentrations of P4 at -4 d than cows with lower intensity estrus or no estrous expression (4.6 ± 0.2 vs. 3.6 ± 0.2 vs. 3.7 ± 0.2 ng/mL). Cows with greater concentrations of P4 at -4 d had greater P/AI (32.8 ± 4.4 vs. 22.4 ± 4.5%), whereas cows with lesser concentrations of P4 at d0 for either timed AI (35.2 ± 3.4 vs. 19.6 ± 4.6%) or spontaneous estrus (31.8 ± 2.8 vs. 23.4 ± 3.2%) had greater P/AI. Cows with greater concentrations of P4 7 d post-timed AI had greater P/AI compared with cows that had lower concentration of P4 (39.1 ± 2.9 vs. 24.7 ± 2.6%). Similarly, cows that had lower concentrations of P4 at 7 d, 14 d and 21 d post-spontaneous estrus tended to have lower P/AI when compared with cows with greater concentrations of P4. Overall, concentrations of P4 prior to and at AI were associated with greater estrous intensity and P/AI at both spontaneous and timed AI events.

Cattle adapted to tropical and subtropical environments: genetic and reproductive considerations.

Efforts to understand biological functions and develop management schemes specific to Bos indicus-influenced cattle raised in tropical and subtropical environments are critical to meet the increasing global demand for protein. In the United States, B. indicus breeds are mostly used to generate B. indicus × B. taurus crosses with increased thermal and parasite tolerance, while retaining some productive characteristics of B. taurus cattle. Although crossbreeding represents a proven strategy to improve cattle adaptation almost immediately, research has also attempted to identify B. taurus genetics that can withstand subtropical and tropical climates. Reduced milk production and delayed reproductive maturation appear to be related with tropical adaptation of B. taurus breeds, as a means to conserve energy under stressful conditions and limited nutrition. Moreover, longevity may be the ultimate adaptation response to unfavorable environments, and retention of bulls and heifers from proven cows is the recommended strategy to improve longevity in B. indicus-influenced herds. Besides selection for longevity, other aspects should be considered when planning reproductive management in tropical and subtropical regions. Bos indicus and B. taurus breeds have multiple differences pertaining to reproductive function, including age at puberty, ovarian dynamics, and pregnancy development. Nutritional strategies such as the stair-step regimen, and use of exogenous progesterone (P4) inserts are options to hasten puberty attainment of late-maturing B. indicus-influenced heifers. Yet, limited pharmacological alternatives are available for reproductive management of B. indicus-influenced females in the United States, which rely on GnRH-based protocols not specifically designed to the reproductive function of B. indicus breeds. In contrast, hormonal protocols based on exogenous P4, estradiol esters, and equine chorionic gonadotropin are available for use in B. indicus females in South America. These include protocols tailored to prepubertal heifers, anestrous cows, and cycling nulliparous or parous females, which often yield pregnancy rates of 50% to fixed-time artificial insemination. The global dairy industry also faces similar challenges in increasing demand and production as the beef industry. Selection of cows capable of sustaining optimal milk yield, reproductive success, and health status in hot and humid conditions is essential for optimal dairy production in subtropical and tropical regions.

Rumen-protected B vitamin complex supplementation during the transition period and early lactation alters endometrium mRNA expression on day 14 of gestation in lactating dairy cows.

Greater metabolic demands in high-producing dairy cows are believed to be a cause of sub-fertility in these animals. Previously, supplementation with vitamin B complex molecules has shown benefits in improving milk production, health, and reproductive efficiency of dairy cows. The primary aim of this project was to determine the effects of rumen-protected vitamin B complex supplementation of 100 g of Transition VB (Jefo, St. Hyacinthe, QC, Canada) and 4 g of Lactation VB (VB; Jefo), during the transition and early lactation periods, respectively, compared with a control diet containing no supplementation on d 14 endometrial outcomes of pregnancy. In the vitamin B supplemented cows, we expect to see a change in the mark-up of endometrial genes important for embryo survival before implantation. Multiparous Holstein cows were enrolled into the study 3 wk before parturition and were randomly assigned to either the VB or control treatment. Twice-a-week blood samples, weekly milk samples, and daily feed intake were collected. Cows were enrolled onto a double-ovsynch protocol at 33 ± 3 d postpartum and inseminated by timed artificial insemination. Milk production and components, concentrations of BHB, haptoglobin, and progesterone in serum, and ovarian dynamics were also measured, but no treatment effect was observed. The uterus was flushed on d 14 after artificial insemination (around 72 DIM) for conceptus collection, and endometrial samples were collected at the same time. Overall, 42 cows were flushed and 13 embryos were collected. Analysis of mRNA expression of genes related to embryo development, immune system, adhesion, and regulation of vitamin B molecules showed that OXTR, MUC5B, MUC1, IL1B, SPP, TRD, FZD8, and FOLR1 genes were significantly upregulated in the VB group. Vitamin B supplementation had no effect on the size of the embryo and ovulatory follicle or corpus luteum diameter at embryo collection. In conclusion, the benefits of strategic dietary VB supplementation during the transition and early lactation might be directly linked to endometrial functions required for embryo survival during the peri-implantation period.

Effect of estrous expression on timing and failure of ovulation of Holstein dairy cows using automated activity monitors.

The aim of this study was to determine if estrous expression, as measured by an automated activity monitor (AAM), affects timing and failure of ovulation of lactating Holstein dairy cows. Cows were equipped with 2 AAM, 1 neck-mounted (AAMC) and 1 leg-mounted (AAML), by 10 d postpartum and enrolled into the trial when their activity crossed the alert threshold on the AAMC. A total of 850 episodes of estrus from 293 different cows were used for this study. When cows were enrolled, their ovaries were scanned by transrectal ultrasonography and gait and body condition scored. Ovaries of cows detected in estrus were scanned twice daily for a maximum of 3 d to determine the disappearance of the preovulatory follicle (ovulation) and the interval from estrus to ovulation was calculated. Physical activity data recorded from the AAM were used to determine estrus behavior using 2 traits: (1) peak activity and (2) duration. Peak activity was only available for the AAML. Peak activity was defined as the maximum activity during an estrus episode. Duration of estrus was defined as the time the activity of the cow exceeded threshold values set by the AAM software. The AAMC correctly identified 87.8% of the estrus alerts, with 12.2% false positives. The average (±standard deviation) intervals from activity alert to ovulation were 25.8 ± 10.2 and 24.7 ± 9.3 h for the AAMC and AAML, respectively. Changes in estrous expression were associated with differences in the interval from alert to ovulation. Cows with short intervals to ovulation were found to have less intense estrous expression than cows with medium and long length intervals to ovulation using the AAMC, whereas using the AAML, cows with short intervals to ovulation exhibited less intense estrous expression than cows with medium but the same as those with long intervals to ovulation. Furthermore, irrespective of the AAM, estrus events with less estrous expression had increased odds of having a short interval to ovulation (below the median of 20 h) when compared with those having greater estrous expression (2.6 and 1.9 increased odds for the AAMC and AAML, respectively). Ovulation failure was affected by estrous expression because estrus events with greater peak activity or longer duration had reduced ovulation failure compared with those with less estrous expression (AAMC peak activity: 1.9 ± 1.4 vs. 9.5 ± 1.7%; AAML peak activity: 2.3 ± 1.4 vs. 6.2 ± 1.5%; AAMC duration: 2.1 ± 1.4 vs. 8.9 ± 1.7%). In addition, cows with more estrous expression had greater pregnancy per artificial insemination than those with less estrous expression with both the AAMC (42.3 ± 0.4 vs. 31.7 ± 0.4%) and the AAML (43.1 ± 0.4 vs. 36.3 ± 0.4%). Pregnancy per artificial insemination results were consistent even when removing cows that failed to ovulate. In conclusion, expression of estrus was highly associated with ovulation timing, ovulation failure, and fertility when using 2 different AAM. Cows with greater estrous expression have longer intervals from activity alert to ovulation, experience less ovulation failure, and have greater pregnancy per artificial insemination.

Effect of metritis on endometrium tissue transcriptome during puerperium in Holstein lactating cows.

The objective of this prospective cohort study was to evaluate the effect of parity and uterine health status postpartum on the gene expression profile of the endometrium early post-partum. Twenty-four Holstein cows were randomly selected (16 multiparous (MP) and 8 primiparous (PP)) and endometrium biopsies were collected on days 1, 3, and 6 after calving and clinically monitored for metritis. Rectal temperature was measured twice and fever was defined as a temperature ≥39.5 °C. A case of metritis was diagnosed with the presence of red-brown watery, foul-smelling uterine discharge or a purulent discharge with more than 50% pus and fever between days 1 and 6 postpartum. Cows were then retrospectively selected (cows diagnosed with metritis were paired with healthy ones) to analyze the expression of 66 genes measured on the NanoString nCounter Analysis System. The genes selected were related with adhesion, immune system, steroid and prostaglandin biosynthesis regulation, insulin metabolism and transcription factors, and nutrient transporters. The results indicated a different pattern on genes related to immune function by parity. PTX3, involved in antigen presentation, was increased in healthy MP compared with healthy PP whereas inflammatory cytokine TNFα and complement-related protein SERPING1 was upregulated in MP compared with PP (P < 0.05). As expected, presence of a metritis condition affected the expression of genes related to immune function. There was an increased expression of the antiviral factor MX2 and MYH10 gene, which is involved in macrophages recruitment, in metritic compared with healthy cows (P < 0.05). Differences in uterine involution from cows diagnosed with metritis were reflected by the downregulation of IGF1 (P < 0.10), involved in endometrium remodeling, and a possible compensatory upregulation of its receptor IGFR1 (P < 0.05). A greater expression of prostaglandins and oxytocin receptors (PGR and OXTR), involved in the involution process, were observed in metritic PP compared with healthy PP (P < 0.05). Overall, it seems that metritis significantly modulate processes closely tied with the physical involution of the uterus early post-partum (IGF1, IGFR1, PGR, OXTR), whereas both metritis and multiparous cows tended to upregulate genes related to immune response (PTX3, TNFα, SERPING1, MX2, MYH10).

Supplementing Ca salts of soybean oil after artificial insemination increases pregnancy success in Bos taurus beef cows.

Two experiments investigated the effects of supplementing Ca salts of soybean oil (CSSO) during early gestation on reproductive function and pregnancy rates to AI in Bos taurus beef cows. In Exp. 1, 771 suckled, lactating, multiparous Angus cows were divided into 22 groups of approximately 35 cows per group and timed inseminated on day 0. After AI, groups were assigned randomly to receive (as-fed basis) 100 g of ground corn + 100 g of soybean meal per cow/d, in addition to 1) 100 g/cow daily of CSSO (n = 11) or 2) 87 g of prilled saturated fat + 13 g of limestone per cow/d (CON; n = 11). Groups were maintained in individual tall fescue-dominated pastures and offered treatments from day 0 to 21. Pregnancy status was determined between days 45 and 55 via transrectal ultrasonography. Cows receiving CSSO had greater (P = 0.01) pregnancy rates to timed AI compared with CON (60.2 vs. 51.7%; SEM = 4.2). In Exp. 2, 90 suckled, lactating, multiparous Angus × Hereford cows housed in 18 drylot pens (5 cows per pen) were assigned to the same timed AI program and treatments from Exp. 1 (9 pens per treatment) and received 20 kg/d (DM basis) of grass-alfalfa hay. Transrectal ultrasonography was performed to verify ovulation and corpus luteum (CL) volume before AI (day 0), on days 7 and 15. After ultrasonography on day 15, cows diagnosed without a CL on day 0, but with a CL greater than 0.38 cm3 in volume on days 7 and 15 (2 or 3 cows per pen; CSSO, n = 20; CON, n = 24), were assigned to conceptus collection via transcervical flushing and endometrial biopsy in the uterine horn ipsilateral to the CL. Blood samples were collected for FA analysis on days 0, 7, and 15. Blood was collected from cows not assigned to conceptus collection for whole-blood RNA extraction on day 20 and for pregnancy diagnosis on day 30 by measuring concentrations of pregnancy-associated glycoproteins. Cows receiving CSSO had greater (P ≤ 0.04) mean plasma concentrations of linoleic acid and ω-6 FA compared with CON on days 7 and 15. Moreover, CSSO supplementation increased (P = 0.05) mRNA expression of interferon-tau by the conceptus and blood mRNA expression of interferon-stimulated gene 15 and 20,50-oligoadenylate synthetase on day 20 in gestating cows. Hence, post-AI CSSO supplementation to B. taurus beef cows improved pregnancy rates to timed AI, which can be associated with increased mRNA expression of interferon-tau by the conceptus when CSSO is supplemented during early gestation.

Integrating an automated activity monitor into an artificial insemination program and the associated risk factors affecting reproductive performance of dairy cows.

The aim of this study was to compare 2 reproductive programs for the management of first postpartum artificial insemination (AI) based on activity monitors and timed AI, as well as to determine the effect of health-related factors on detection and expression of estrus. Lactating Holstein cows (n = 918) from 2 commercial farms were enrolled. Estrous cycles of all cows were presynchronized with 2 injections of PGF2α administered 2 wk apart. Treatments were (1) first insemination performed by timed AI (TAI) and (2) first insemination based upon the detection of estrus by activity monitors (ACT; Heatime, SCR Engineering, Netanya, Israel) after the presynchronization, whereas cows not inseminated by the detection of estrus were enrolled in the Ovsynch protocol. Body condition score (BCS; scale 1 to 5), hock score (scale: 1 to 4), gait score (scale: 1 to 4), and corpus luteum presence detected by ovarian ultrasonography were recorded twice during the presynchronization. On the ACT treatment, 50.5% of cows were inseminated based on detected estrus, whereas 83.2% of the cows on the TAI treatment were inseminated appropriately after the timed AI protocol. Pregnancy per AI did not differ by treatment (30.8 vs. 33.5% for ACT and TAI, respectively). Success of pregnancy was affected by parity, cyclicity, BCS, milk production, and a tendency for leg health. In addition, treatment × cyclicity and treatment × parity interactions were found to affect pregnancy success, where anovulatory cows and older cows had compromised pregnancy outcomes on the ACT treatment but not on the TAI treatment. Factors affecting pregnancy outcomes varied among farms. Hazard of pregnancy by 300 DIM was affected by farm, parity, BCS, a treatment × cyclicity interaction, and a tendency for an interaction between leg health and farm. Detection of estrus was affected by farm, parity, cyclicity, and leg health, but not BCS or milk production. Expression of estrus was compromised in anovular and older cows, and by the timing of the estrus event, but not by gait score, BCS, or milk production. Increased duration of estrus, but not intensity of estrus, improved pregnancy per AI. In conclusion, using an automated activity monitor for the detection of estrus within a Presynch-Ovsynch program resulted in similar pregnancy per AI and days open compared with a reproduction program that was strictly based on timed AI for first postpartum AI. In contrast, notable variations in reproductive outcomes were detected between farms, suggesting that the use of automated activity monitors is prone to individual farm management.

Period of dominance of the ovulatory follicle influences embryo quality in lactating dairy cows.

Length of dominance of the ovulatory follicle and exposure to oestradiol (OE(2)) during proestrus can affect fertility. Lactating cows had their oestrous cycle pre-synchronized and were subjected to one of the four synchronization treatments. Cows in the oestrus detection (OD) treatment received GnRH on day 6 of the oestrous cycle, PGF(2alpha) 7 days later, and were inseminated at detected oestrus. The remaining cows were subjected to the Ovsynch (OVS) protocol (day 0 GnRH, day 7 PGF(2alpha), day 9 GnRH, and timed artificial insemination (AI) 12 h later) starting on day 3 (OVS3) or day 6 (OVS6 and OVS6E) of the oestrous cycle. Cows in the OVS6E treatment received an injection of 0.5 mg oestradiol cypionate 36 h before AI. Ovaries were examined by ultrasonography and blood was sampled for progesterone and OE(2) concentrations. Uteri were flushed 6 days after AI and recovered embryos-oocytes evaluated. Diameter of the ovulatory follicle at AI differed (P<0.01) among treatments, and it was the largest for OVS3 cows, which also had extended (P<0.01) length of follicular dominance. During proestrus, OD and OVS6E cows had increased (P<0.01) OE(2) concentrations. Fertilization was not altered by treatments, and maximum fertilization was achieved when the number of accessory spermatozoa was >7. Proportions of viable embryos in relation to embryos and embryos-oocytes recovered were smaller for OVS3 cows (P<0.01) than the other treatments, and embryos from OVS3 cows also had fewer (P<0.01) blastomeres and tended (P=0.09) to have a lower proportion of live blastomeres. Extending the period of follicle dominance did not alter fertilization but reduced (P<0.001) embryo quality. Embryo quality was compromised even when the dominance of the ovulatory follicle was extended by only 1.5 days.

Efficacy of an injection of dinoprost tromethamine when given subcutaneously on luteal regression in lactating Holstein cows.

The objectives of these studies were to evaluate the efficacy of a PGF(2alpha) (PGF) analog given through different routes on causing luteal regression in lactating dairy cows. In Experiment 1, lactating Holstein cows (n=118) at random stages of lactation were blocked by parity and days in milk (DIM) and, within each block, randomly assigned to receive PGF as an intra-muscular (IM) injection in the semimembranous/semitendinous muscle (CON), subcutaneous (SC) injection in the cervical area (SCN), or SC injection in the ischio-rectal fossa (IRF). Blood was sampled at 0, 12, 24, 36, and 48 h after treatment for assessment of progesterone concentration. In Experiment 2, a total of 379 lactating Holstein cows, 46+/-7 DIM, were blocked by DIM and, within each block, randomly assigned to receive treatment similar to CON or IRF groups from Experiment 1. Blood was sampled 0 and 48 h after treatment for assessment of progesterone concentration. Cows were classified as experiencing luteal regression when progesterone concentration was <1.0 ng/mL or <40% of initial concentration (0 h=100%). In Experiment 1, there was no effect of route of PGF treatment on decline in progesterone concentration and on the proportion of cows experiencing luteal regression by 12, 24, 36, and 48 h after treatment. Similarly, in Experiment 2, route of treatment did not affect either the decline in progesterone concentration or the proportion of cows that had luteal regression by 48 h after treatment. Treatment of lactating dairy cows with 25mg of PGF given SC in the ischio-rectal fossa did not affect either the decline in progesterone concentration or the proportion of cows that experienced luteal regression by 12, 24, 36, and 48 h after PGF treatment.

Effect of resynchronization with GnRH on day 21 after artificial insemination on pregnancy rate and pregnancy loss in lactating dairy cows.

The objectives of the present study were to determine the effects of resynchronization with GnRH on Day 21 after artificial insemination (AI) on pregnancy rate and losses of pregnancy in lactating dairy cows. Holstein cows (n=585) on two dairy farms were assigned to one of two treatments in a randomized complete block design. On Day 21 after a pre-enrollment AI, animals assigned to the resynchronization (RES) group received 100 microg of GnRH i.m., whereas animals in the control (CON) group received no treatment. All animals were examined ultrasonographically on Days 21 and 28 after AI, and blood samples were taken for progesterone measurement on Day 21. Pregnancy was diagnosed on Day 28 and reconfirmed 14 days later. Nonpregnant cows on Day 28 were inseminated using timed AI after the completion of the Ovsynch protocol 10 and 17 days after enrollment in the study for RES and CON groups, respectively. Progesterone concentration > or =2.35 ng/ml was used as an indicator of pregnancy on Day 21. For RES and CON cows, pregnancy rate at Days 21 (70.9% versus 73.0%, P<0.56), 28 (33.1% versus 33.6%; P<0.80) and 42 (27.0% versus 26.8%; P<0.98) after the pre-enrollment AI did not differ. Administration of GnRH on Day 21 after AI had no effect on pregnancy loss in RES and CON groups from days 21 to 28 (53.2% versus 53.5%; P<0.94) and days 28 to 42 (17.9%; P<0.74) after AI. Pregnancy rate after the resynchronization period was similar for both treatment groups. Resynchronization with GnRH given on Day 21 after AI for initiation of a timed AI protocol prior to pregnancy diagnosis does not affect pregnancy rate and pregnancy loss in lactating dairy cows.