Reduced embryo yield obtained from superstimulated ewes with low circulating AMH concentration is improved by lengthening the FSH treatment.

The aim of the present study was to evaluate: 1) the association between AMH, AFC, superovulatory response and embryo yield in sheep; and 2) the effect of FSH treatment length during superstimulation of the first follicular wave on ovarian response and embryo yield, particularly in ewes with low and high AMH. The experiment was performed on 63 Polled Dorset ewes that received an ovarian superstimulatory treatment during the first follicular wave (Day 0 protocol). Ewes were administered a total dose of 240 mg of FSH distributed in six (6-dose regimen, n = 30) or eight (8-dose regimen, n = 33) decreasing doses administered 12 h apart. On Day -9 (random stage of the estrous cycle) and Day 0 (day of the first FSH dose) ovarian ultrasonography was performed and blood samples were collected for AFC and AMH determinations, respectively. A weak positive correlation between AMH and small AFC (follicles <4 mm) was observed (r = 0.23; P = 0.07), and AMH concentration was positively correlated (r = 0.29; P < 0.05) with the number of corpora lutea (CL) determined at embryo collection (i.e., 6 d after insemination). The length of FSH treatment tended (P = 0.06) to affect the ovarian response, such that the number of CL was greater in 8-dose than 6-dose treated ewes, while no differences (P > 0.10) in embryo yield outcomes were observed. For further analysis, ewes were classified into low (<7 ng/mL) and high (>10 ng/mL) serum AMH. In high AMH ewes, there were no differences (P > 0.05) in the number of CL nor embryo yield between the 6-dose and 8-dose treatment (e.g., 7.8 ± 2.4 and 8.3 ± 2.5 transferable embryos, respectively; P = 0.92). Conversely, for low AMH ewes, fertilized ova and embryo yield were greater (P ≤ 0.05) for ewes receiving the 8-dose than the 6-dose superstimulatory treatment (e.g., 8.4 ± 2.8 vs. 2.7 ± 0.9 transferable embryos, respectively, P ≤ 0.05). In conclusion, embryo production in poor responding ewes with low low circulating AMH is improved by extending the superstimulatory treatment length from 6 to 8 FSH doses.

GnRH dose at initiation of a 5-day CO-Synch + P4 for fixed time artificial insemination in suckled beef cows.

The objective of the present study was to evaluate the effect of GnRH dose administered at initiation (GnRH-1) of a 5-day CO-Synch + P4 protocol on ovulatory response, expression of estrus, and fertility in suckled beef cows. Suckled beef cows (n = 1101) at four locations were randomized to receive either 100 or 200 µg of gonadorelin acetate at initiation (D-8) of a 5-day CO-Synch + P4 protocol concurrently with insertion of an intravaginal progesterone (P4) device. On D-3 the P4 device was removed, two doses of prostaglandin F2α were administered concurrently and a patch was applied to evaluate expression of estrus. Artificial insemination was performed 72 h after P4 device removal (D0) simultaneously with the administration of 100 µg of gonadorelin acetate (GnRH-2). Increasing GnRH dose at initiation of a 5-day CO-Synch + P4 did not enhance ovulatory response (P = 0.57) to GnRH-1, expression of estrus (P = 0.79), nor pregnancies per AI (P/AI; P = 0.91). Both follicle size (quadratic) and circulating P4 (linear) affected (P < 0.01) ovulatory response to GnRH-1 independent of dose. Cows that had ovulation to GnRH-1 had smaller (P < 0.001) follicle size on D-3 and reduced (P = 0.05) expression of estrus compared to cows that did not have ovulation to GnRH-1, however, P/AI did not differ (P = 0.75). In conclusion, increasing the dose of GnRH-1 in the 5-day CO-Synch + P4 protocol did not enhance ovulatory response, expression of estrus, or P/AI in suckled beef cows.

Effect of timing of prepartum vaccination relative to pen change with an acidogenic diet on lying time and metabolic profile in Holstein dairy cows.

The objective was to assess the effect of prepartum vaccination timing relative to pen change with an acidogenic diet at 28 or 21 d before expected parturition (dpp) on lying time (LT), prepartum serum energy status (glucose, IGF-1, and nonesterified fatty acids), urine pH, and serum Ca at calving in pregnant Holstein dairy cows. Pregnant multiparous Holstein cows (n = 308) from 1 large dairy herd were randomly allocated into 1 of 3 treatment groups at 35 ± 3 dpp as follows: (1) vaccination at 28 dpp and pen change at 21 dpp (V28PC21; n = 108), (2) vaccination and pen change at 28 dpp (V28PC28; n = 99), and (3) vaccination and pen change at 21 dpp (V21PC21; n = 101). When cows changed pens, an acidogenic diet was introduced. Every other week, a group of 43 to 53 animals were enrolled and electronic data loggers (IceQube, IceRobotics) were fitted to the hind leg of individual cows to assess their LT. Blood samples were collected at 28, 26, 21, 19, 14 dpp and at calving. Parity, body condition score, days dry, and gestation length were not different among groups. Overall, V28PC28 cows had 7 additional days in prepartum pens consuming an acidogenic diet compared with V28PC21 or V21PC21 cows. Regardless of treatment group, cows in the far-off pen had 43 min/d less LT (709 vs. 753 min/d) and increased day-to-day coefficient of variation of LT (0.21 vs. 0.10) compared with cows within the prepartum pen. On average, for the 7 d following vaccination alone (28 to 22 dpp period), V28PC21 cows had ~22 min/d less LT compared with V21PC21 cows. Serum concentrations of glucose, nonesterified fatty acids, and IGF-1 were altered following vaccination alone, pen change alone, or vaccination plus pen change with an acidogenic diet before calving. At calving, V28PC21 cows had greater glucose concentrations (6.45 mmol/L) compared with V21PC21 cows (5.76 mmol/L), with V28PC28 cows intermediate (6.11 mmol/L). The assessment of Ca status at calving revealed that V28PC21 cows had greater Ca concentration (2.34 mmol/L) with lower subclinical hypocalcemia (<2.0 mmol/L; 17.3%) compared with V21PC21 cows (2.17 mmol/L and 31.9%), with V28PC28 cows intermediate (2.28 mmol/L and 25.2%). Serum concentrations of IGF-1 at calving were also greater for V28PC21 (3.43 nmol/L) cows compared with V21PC21 (2.69 nmol/L), with V28PC28 cows intermediate (3.07 nmol/L). Overall, V28PC21 cows had greater serum glucose, IGF-1, and ~46% reduction in subclinical hypocalcemia (from 31.9 to 17.3%) compared with V21PC21 cows but did not differ from V28PC28 cows (25.2%). These findings provided evidence that vaccinating cows at 28 dpp, followed 7 d later by pen change with an acidogenic diet at 21 dpp, would be beneficial.

Effects of prepartum vaccination timing relative to pen change with an acidogenic diet on serum and colostrum immunoglobulins in Holstein dairy cows.

The objective of the present study was to assess the effects of prepartum vaccination timing relative to pen change with an acidogenic diet at 28 or 21 d before expected parturition (dpp) on colostral and serum IgG concentrations at calving in pregnant Holstein dairy cows. Pregnant multiparous Holstein cows (n = 308) from one large dairy herd were randomly allocated into 1 of 3 treatment groups at 35 ± 3 dpp: (1) vaccination at 28 dpp and pen change at 21 dpp (V28PC21; n = 108), (2) vaccination and pen change at 28 dpp (V28PC28; n = 99), and (3) vaccination and pen change at 21 dpp (V21PC21; n = 101). An acidogenic diet was fed when cows changed pens at 28 or 21 dpp. Blood and colostral samples were collected within 1 h following parturition. The total number of clinical mastitis (CM) cases within the first 150 d in milk (DIM) were recorded. The V28PC21 cows had greater colostral IgG concentrations at calving (160.4 ± 7.0 g/L) compared with V21PC21 cows (134.4 ± 7.0 g/L), and V28PC28 cows were intermediate (148.3 ± 7.2 g/L). At calving, V28PC21 cows had lower serum IgG concentrations (29.1 ± 1.2 g/L) compared with V21PC21 cows (32.2 ± 1.2 g/L) or V28PC28 cows (32.6 ± 1.3 g/L). Overall, 41% of V21PC21 cows received the booster vaccinations with at least 21 d before actual calving compared with V28PC21 or V28PC28 cows (88 and 86% respectively). The shorter the interval from prepartum booster vaccination to calving, the lower the colostral IgG at calving, regardless of treatment groups. Vaccinating at 28 dpp and pen change with an acidogenic diet at 21 dpp tended to reduce the rate of CM within the first 150 DIM compared with V21PC21. These findings provide evidence that vaccinating cows at 28 dpp, followed by pen change with an acidogenic diet at 21 dpp, improved the concentrations of colostral IgG at calving and tended to reduce the rate of CM. The interaction of prepartum vaccination timing relative to feeding an acidogenic diet should be considered when implementing an effective vaccination program to enhance overall herd health.

Association of prepartum lying time with nonesterified fatty acids and stillbirth in prepartum dairy heifers and cows.

The objective of the present study was to assess the association of prepartum lying time (LT) and the coefficient of variation (CV) of LT within 7 d before calving with prepartum serum nonesterified fatty acid (NEFA) concentration at 7 ± 3 d prepartum (dpp) and stillbirth. Prepartum pregnant Holstein heifers and cows from 3 dairy herds were used (n = 1,044). Animals were housed in freestall barns using a prepartum pen 21 d before the expected calving date and were moved into a contiguous maternity pen at parturition. Monthly, cohorts of 20 to 36 animals (heifers and cows combined) were enrolled at each farm and electronic data loggers (IceQube, IceRobotics, Edinburgh, UK) were fitted to the hind leg of individual animals to assess their behavioral activity. Stillbirth was defined as a calf born dead or died during the first 24 h after parturition in dams with normal gestation length. The LT was recorded for the last 7 dpp to assess differences among dams with stillbirth versus those with a calf born alive. Mean LT within 7 d before blood NEFA collection was assessed to determine the association with prepartum serum NEFA at 7 ± 3 dpp. Blood samples for the assessment of serum NEFA concentration were collected from prepartum animals at 14 ± 3 and at 7 ± 3 dpp. Blood samples for total serum calcium concentration were collected from postpartum cows within 48 h after parturition to assess differences among cows with stillbirth versus those with a calf born alive. Data were analyzed using CORR, MIXED, or GLIMMIX procedures of SAS (SAS Institute Inc., Cary, NC). Dams experiencing dystocic births had a greater proportion of stillbirth, but herd, parity, and season did not have an effect. Dams with a stillborn calf had reduced LT and increased CV of LT within the last 7 dpp compared with dams with a calf born alive, regardless of parity. Multiparous cows with a stillborn calf had higher prepartum serum NEFA concentration compared with multiparous cows with a calf born alive, but this did not differ for first-calf heifers. Regardless of parity, the proportion of postpartum cows with hypocalcemia was higher for dams with a stillborn calf compared with those with a calf born alive. Regardless of parity, LT of prepartum dams was strongly correlated with the CV of LT (as LT increased, the CV decreased), and prepartum dams with a mean LT between 11 and 15 h/d had the lowest serum NEFA concentration compared with dams with LT of 8 to 10 or >16 h/d. Serum NEFA concentrations at 7 ± 3 dpp was positively correlated with the CV of LT within 7 d before blood sample. These results show that the dam's prepartum LT and its consistency over time are associated with prepartum serum NEFA and calf survival at calving.

Optimization of a 5-day fixed-time embryo transfer (FTET) protocol in heifers I. Manipulation of circulating progesterone through reutilization of intravaginal progesterone devices during FTET.

The objectives of the present study were to: 1) compare the reproductive efficiency of embryo transfer (ET) recipients after synchronization of estrus or a 5-day synchronization of ovulation protocol for fixed time ET (FTET), and 2) determine the effect of reutilization of intravaginal P4 devices (CIDRs), up to four times, in a 5-day FTET protocol. In Experiment 1, 817 dairy heifers were assigned to one of three groups: PGF + estrus detection, 5-d FTET protocol with new (1.38 g P4) or 2nd use CIDR (previously used once for 5 d). Fresh in vitro produced embryos were transferred 7 ± 1 day after estrus (PGF + estrus) or GnRH (5-day FTET). Utilization rate (transferred/treated) was greater (P < 0.001) in heifers submitted to FTET compared to ET after estrus, however pregnancies per ET (P/ET) were not different (P > 0.10). As a result, pregnancy per treated (P/treated) recipient was greater (P < 0.05) in heifers in the 5-day FTET protocol. In Experiment 2, 40 dairy heifers without a corpus luteum (CL) were randomly allocated into one of four groups using new, 2nd use, 3rd use (previously used twice for 5 d/each), or 4th use (previously used thrice for 5 d/each) CIDRs. Circulating P4 was reduced (P < 0.01) with each reutilization. In Experiment 3, ovarian follicular dynamics were evaluated in 238 dairy heifers submitted to a 5-day protocol with either new, 2nd use, 3rd use or 4th use CIDRs at random stages of the estrous cycle. Prostaglandin F2α (PGF) was administered at CIDR removal and again 24 h later. Ovulation was induced by GnRH treatment 72 h after CIDR removal. Preovulatory follicle diameter increased (P < 0.001) progressively with increasing CIDR reutilization. Ovulation rate did not differ between treatments, however, interval from CIDR removal to ovulation decreased (P < 0.001) in heifers receiving 3rd and 4th use CIDRs compared to new or 2nd use. Finally, in Experiments 4 and 5, 1203 heifers submitted to a 5-day FTET protocol were randomly assigned to receive either a new CIDR, a 3rd use CIDR (Experiment 4) or a 4th use CIDR (Experiment 5). Despite the increase in CL volume on D5 in heifers treated with 3rd use (P = 0.03) or 4th use CIDRs (P < 0.01), there were no differences (P > 0.05) in utilization rate, P/ET, or P/treated. Thus, use of a 5-day FTET synchronization protocol improves reproductive efficiency by increasing recipient utilization, and reutilization of CIDRs up to four times in recipient dairy heifers does not compromise reproductive performance.

Quantifying the effects of mastitis on the reproductive performance of dairy cows: A meta-analysis.

The objective of this study was to conduct a meta-analysis to quantitatively characterize the effects of mastitis on the reproductive performance of dairy cows as well as to identify factors that interact with this relationship. A total of 29 publications were identified that contributed 24, 41, 27, 38, and 13 trial results to the meta-analysis of how mastitis is related to time to first service (TFS), days open (DO), services per conception (SPC), pregnancies per insemination at first service (FSP/AI) and pregnancy loss (PL), respectively. The meta-analyses were conducted using multilevel linear mixed-effects models. Overall, high levels of heterogeneity were present and meta-regression models only explained a small amount of heterogeneity. Results suggest that cows with mastitis pre-first insemination experience, on average, an additional 13.29 d to first service [95% confidence interval (CI): 6.64, 19.95] when compared with cows with no mastitis in the same time period. Moreover, in relation to cows with no mastitis in the same time period, 22.34 additional DO (95% CI: 12.89, 31.79) were estimated, on average, for cows with clinical mastitis at pre-insemination leading to conception. Additionally, 32.41 added DO (95% CI: 20.58, 44.25) were estimated, on average, for cows with clinical mastitis at pre- or post-insemination leading to conception compared with cows with no mastitis in the same time period. Finally, 20.03 additional DO (95% CI: 3.11, 36.95) were estimated, on average, for cows with subclinical mastitis pre- or post-insemination leading to conception compared with cows with no mastitis in the same time period. Effect size estimates from the meta-regression models for SPC, in relation to cows with no mastitis in the same time period, suggest that, on average, SPC increases by 0.46 inseminations (95% CI: 0.30, 0.62) for a cow experiencing mastitis pre-insemination leading to conception. When mastitis occurs either pre- or post-insemination leading to conception, SPC is expected to increase, on average, by 0.72 inseminations (95% CI: 0.48, 0.95) compared with cows with no mastitis in the same time period. The estimated effect sizes for FSP/AI suggest a risk ratio of conceiving at first insemination of 0.90 (95% CI: 0.82, 0.99) for cows with mastitis diagnosed pre-first insemination with respect to cows with no mastitis in the same time period, and a risk ratio of 0.79 (95% CI: 0.72, 0.86) for cows diagnosed with mastitis either pre- or post-first insemination with respect to cows with no mastitis in the same time period. Publication bias was identified in 4 of the meta-analysis models (TFS, DO, SPC, and FSCR), but no influential trials were identified in any models; the reliability of the meta-analysis results should be interpreted carefully keeping these limitations in mind. Further meta-regression analysis would be valuable as additional studies are published that report other potential sources of heterogeneity.

Lengthened superstimulatory treatment in cattle: Evidence for rescue of follicles within a wave rather than continuous recruitment of new follicles.

A study was designed to compare the effects of a conventional (4 days) versus a lengthened (7 days) superstimulation protocol on follicle dynamics and to test the hypothesis that superstimulatory treatment only rescues small follicles within the wave. Nonlactating beef cows received a progesterone-releasing intravaginal device [PRID] and were superstimulated with 400-mg FSH on the day of follicle ablation-induced wave emergence (Day 0). The control group (n = 5) received FSH over 4 days, whereas the long group (n = 5) received FSH over 7 days. PGF was administered twice on Day 2 (control group) or 5 (long group), and PRIDs were removed 24 hours after the first PGF. Cows received 25-mg LH 24 hours after PRID removal. The cows chosen for the present study represented a subset from a larger group of 24 cows in which superovulation results were obtained and published. Cows in the present study were those with the lowest antral follicle counts at the time of wave emergence in order to facilitate tracking of individual follicles. Daily ultrasonographic examinations monitored follicle diameters and numbers. A reduction (P < 0.01) in the number of 1- to 2-mm follicles from Day 0 to the end of the superstimulatory treatment was associated with a progressive shift to the next size category in both groups. On the day of LH treatment, the number of follicles in any size category did not differ between groups. The number of follicles of diameter 5 mm or less decreased during superstimulation (i.e., no continuous recruitment of small follicles; P < 0.001), and the number of follicles of diameter 1 mm or greater at the end of superstimulation did not differ from that at the beginning of superstimulation (P = 0.739). However, the total number of follicles of diameter 3 mm or greater at the end was greater than the number of follicles of diameter 3 mm or greater at the beginning of superstimulation because of growth of the 1- to 2-mm population during treatment (P < 0.001). Follicle growth was slower in the long group than in the control group. Results support the hypothesis that both 4-day and 7-day superstimulatory protocols rescue small antral follicles present at the time of wave emergence; there was no evidence for continuous recruitment of new follicles. Results also provide rationale for the hypothesis that a lengthened treatment protocol is associated with greater follicle maturation and capacity to ovulate.

Sensitivity of a real-time polymerase chain reaction for Tritrichomonas fetus in direct individual and pooled preputial samples.

The objective of this study was to evaluate the sensitivity of a commercially available real-time polymerase chain reaction (PCR) test for the detection of Tritrichomonas fetus in individual and pooled direct preputial samples. Two samples were collected and processed once a week from nine T. fetus-infected bulls (n = 121) and placed into either an InPouch TF or 2 mL of PBS. Preputial samples were also collected into both media and PBS from 1016 other bulls. All pouches were cultured and evaluated as per manufacturer's instructions. The prepuce samples collected directly into PBS were individually evaluated using real-time PCR by a commercial diagnostic laboratory. Direct preputial samples were then randomly divided for pooling into groups of 5 and 10 samples, ensuring that every pool had one sample from a known infected bull before testing using real-time PCR. Sensitivity was estimated for culture and real-time PCR of the 121 direct and culture-enriched individual samples from nine infected bulls. There were no differences (P = 0.12) among the sensitivity estimates for culture, 95.0% (95% confidence interval [CI]: 89.6%-97.7%); real-time PCR of culture-enriched samples, 95.9% (95% CI: 90.7-98.2); and direct preputial samples, 90.1% (95% CI: 83.5-94.2). There was also no significant difference (P = 0.08) between the sensitivity of real-time PCR for direct preputial samples in 110 pools of 5 (83.6%, 95% CI: 75.6-89.4) or 10 samples (77.3%, 95% CI: 68.6-84.1). The use of three sequential direct samples, collected in PBS at weekly intervals and tested by real-time PCR, increased the sensitivity to 100% for pools of 5 and 96% for pools of 10. In conclusion, direct preputial samples collected in PBS and tested by real-time PCR individually have comparable sensitivity to culture and real-time PCR in enriched samples. The use of pooled direct preputial samples appears to be relatively sensitive. However, this strategy requires repeated sampling to optimize sensitivity. Real-time PCR testing of preputial samples collected directly into PBS with the option of pooling would decrease the cost associated with screening bulls, and increase the feasibility of large epidemiological studies and active surveillance.

Cow attributes, herd management, and reproductive history events associated with the risk of nonpregnancy in cow-calf herds in Western Canada.

To identify herd management and cow characteristics associated with the reproductive success of cow-calf herds in Western Canada, 33,391 beef cows were followed from the beginning of the breeding season in 2001 through pregnancy testing in 2002. Breeding management and cow-level risk factors such as age, body condition score (BCS), and previous reproductive history, were measured through a series of herd visits by project personnel and records maintained by the herd owner. Pregnancy status was measured in 205 herds in the fall of 2001 and again in 200 herds in the fall of 2002. Cows least likely to be pregnant in the fall of the year were 10 years old or older, exposed to a bull less than 84 days, had a BCS ≤5 of 9 at pregnancy testing, <5 of 9 before calving, and lost condition between calving and the start of the breeding season, or had a prebreeding BCS <5 of 9 with a loss of condition between breeding and pregnancy testing. Other factors identified that decreased the likelihood of pregnancy in at least one of the 2 years included being a heifer or being a cow exposed to breeding after her first calf, and using a single bull on breeding pasture. Cows vaccinated for bovine viral diarrhea virus and infectious bovine rhinotracheitis and bred on community pastures were more likely to be pregnant than cows that were not vaccinated and bred on community pastures. Cows bred on community pastures that were not vaccinated were also less likely to be pregnant than cows that were not on community pastures regardless of vaccination status. Calving-associated events such as twin birth, Cesarean section or malpresentation, problems such as uterine prolapse or retained placentas, abortion or calf death within 1 hour after birth, or calving late after the start of the breeding season, were also associated with fewer pregnancies after accounting for all other factors.

Early life growth trajectories and future risk for overweight.

OBJECTIVE: Standard approaches have found that rapid growth during the first 2 years of life is a risk factor for overweight in later childhood. Our objective was to test whether growth velocity, independent of concurrent size, was associated with overweight using a nonlinear random-effects model that allows for enhanced specifications and estimations. METHODS: Longitudinal data from a birth cohort in Mexico (n=586) were used to estimate growth trajectories over 0-24 months for body mass index (BMI), length and weight using the SuperImposition by Translation and Rotation (SITAR) models. The SITAR models use a nonlinear random-effects model to estimate an average growth curve for BMI, length and weight and each participant's deviation from this curve on three dimensions-size, velocity and timing of peak velocity. We used logistic regression to estimate the association between overweight status at 7-9 years and size, velocity and timing of BMI, length and weight trajectories during 0-24 months. We tested whether any association between velocity and overweight varied by relative size during 0-24 months or birth weight. RESULTS: SITAR models explained the majority of the variance in BMI (73%), height (86%) and weight (85%) between 0-24 months. When analyzed individually, relative BMI/length/weight (size) and BMI/length/weight velocity during 0-24 months were each associated with increased odds of overweight in late childhood. Associations for timing of peak velocity varied by anthropometric measure. However, in the mutually adjusted models, only relative BMI/length/weight (size) remained statistically significant. We found no evidence that any association between velocity and overweight varied by size during 0-24 months or birth weight. CONCLUSIONS: After mutual adjustment, size during 0-24 months of life (as opposed to birth size), but not velocity or timing of peak velocity, was most consistently associated with overweight in later childhood.

Lengthening the superstimulatory treatment protocol increases ovarian response and number of transferable embryos in beef cows.

This study determined if lengthening the superstimulation protocol from 4 to 7 days would result in an increase in the superovulatory response with no adverse effects on oocyte/embryo competence in beef cows. Follicular ablation was performed, a progesterone-releasing intravaginal device (PRID) was inserted, and cows were assigned to one of two treatment groups 5 to 8 days after ovulation: Control (4 days of follicle stimulating hormone (FSH)) or Long (7 days of FSH; n=12 per group). The FSH treatments were initiated 1.5 days later (Day 0). A dose of 400 mg NIH-FSH-P1 (Folltropin-V) was distributed equally over 8 (Control) or 14 (Long) im injections at 12-h intervals. Prostaglandin F2α (PGF) was administered twice, 12 h apart, on Day 2 (Control) or Day 5 (Long), and PRID were removed 12 h after the second PGF. Both groups were given 25 mg pLH (lutropin-V) im 24 h after PRID removal and AI was done 12 and 24 h later. Ova/embryos were collected 7 days after the pLH injection. The mean (±SEM) number of ≥9 mm follicles at the time of first AI did not differ (P=0.24) between groups, but more ovulations (30.9±3.9 vs. 18.3±2.9, P=0.01) and CL (27.2±2.1 vs. 20.8±2.2, P=0.04) occurred in the Long group. A higher proportion of the ≥9 mm follicles ovulated between 12 and 36 h after pLH in the Long group (93 vs. 69%; P=0.001). Although numerically higher in the Long group, mean numbers of total ova/embryos, fertilized ova, transferable or freezable embryos did not differ. In conclusion, a lengthened superstimulatory treatment protocol resulted in more follicles acquiring the capacity to ovulate with an increased number of ovulations, and without a decrease in oocyte/embryo competence.