Tail Chalk Research Articles

Presynchronization with prostaglandin F2α and gonadotropin-releasing hormone simultaneously improved first-service pregnancy per artificial insemination in lactating Holstein cows compared with Presynch-14 when combined with detection of estrus

This study aimed to determine the effect of 2 simple breeding strategies combining artificial insemination (AI) after detection of estrus (AIED) and timed AI (TAI) on first-service fertility in lactating Holstein cows. Weekly, lactating Holstein cows (n = l,049) between 40 and 46 d in milk (DIM) were randomly assigned to initiate 1 of 2 breeding strategies for first service: Presynch-14 and PG+G. Presynch-14 is a presynchronization strategy with 2 PGF2α treatments 14 d apart with the last PGF2α 14 d before the initiation of the Ovsynch protocol. Cows treated with PG+G receive a simpler presynchronization program that uses PGF2α and GnRH simultaneously 7 d before Ovsynch. In both treatments, cows detected in standing estrus by tail chalk at any time ≥55 DIM were inseminated, and treatment was discontinued (n = 525). Cows completing treatment received TAI from 78 to 84 DIM (n = 526). In a subgroup of cows that received TAI, blood was collected (n = 163) to assess circulating concentrations of progesterone, and ultrasonographic evaluations of ovaries were performed on the day of first GnRH of Ovsynch (n = 162) and PGF2α of Ovsynch (n = 122). The proportion of cows that received TAI was greater for PG+G compared with Presynch-14 (63.5 vs. 31.9%), which increased DIM at first service for cows treated with PG+G compared with Presynch-14 (75.5 ± 0.4 vs. 68.7 ± 0.4). For cows receiving TAI, the ovulatory response to first GnRH of Ovsynch (73.8 vs. 48.8%) and the proportion of cows with functional corpora lutea (92.6 vs. 73.1%) were greater for PG+G than Presynch-14. Cows treated with PG+G had greater overall pregnancy per AI (P/AI) 42 ± 7 d after AI (40.2 vs. 33.6%) and calving per AI (32.1 vs. 25.2%) than Presynch-14. For cows receiving AIED, treatment did not affect P/AI 42 ± 7 d after AI. However, for cows receiving TAI, PG+G increased P/AI compared with Presynch-14 (44.6 vs. 35.2%). Overall, cows receiving TAI had greater P/AI 42 ± 7 d after AI (42.5 vs. 31.5%) and calving per AI (34.1 vs. 23.7%) and decreased pregnancy loss (16.8 vs. 25.2%) than cows receiving AIED. In summary, PG+G increased the proportion of cows receiving TAI and the DIM at first service, P/AI, and calving per AI compared with Presynch-14 when both TAI programs were combined with AIED.

Effect of inducing accessory corpus luteum formation with gonadotropin-releasing hormone or human chorionic gonadotropin on the day of embryo transfer on fertility of recipient dairy heifers and lactating cows

The objective was to determine the effect of inducing an accessory corpus luteum (CL) using GnRH or human chorionic gonadotropin (hCG) on the day of in vitro-produced (IVP) embryo transfer (ET) on pregnancy per ET (P/ET) and calving/ET in dairy heifers and lactating cows. Dairy heifers (11–15 mo of age; n = 1,547) and lactating cows (n = 1,480) detected in estrus by tail chalk (d 0) were used as recipients. Before ET, the presence of a CL was evaluated by transrectal palpation from d 6 to 9 of the estrous cycle. Animals with a CL were randomly assigned to receive 1 of 3 treatments immediately before ET: control (no treatment; n = 1,009), GnRH (86 µg of GnRH; n = 1,085) and hCG (2,500 IU; n = 1,069). Embryos were implanted in the uterine horn ipsilateral to the ovary with a CL (fresh IVP embryos, n = 2,544; vitrified IVP embryos n = 545; slow-freezing IVP embryos, n = 74). Pregnancy diagnosis was performed on d 37 ± 3 of gestation by transrectal palpation. Pregnancy loss data and calving records were collected from the dairy farm management software. Treatment did not affect P/ET, calving/ET, or pregnancy loss either overall or within parity. When treatments inducing CL formation were combined (GnRH + hCG), heifers tended to have greater P/ET than controls (67.7 vs. 63.5%, respectively). Yet, calving/ET were similar. Response variables were also analyzed within embryo type and parity. For heifers receiving stage 6 (blastocyst) fresh IVP embryos, hCG had greater P/ET than controls (74.5 vs. 51.1%, respectively). In addition, GnRH tended to have greater P/ET than controls (67.8 vs. 51.2%, respectively). However, calving/ET in heifers receiving blastocyst fresh IVP embryos was similar among treatments. When only stage 7 (expanded blastocyst) fresh IVP embryos were considered, primiparous GnRH cows had greater P/ET (59.3 vs. 47.1%) and calving/ET (48.6 vs. 38.1%) than hCG. Moreover, hCG showed decreased calving/ET compared with controls in primiparous cows transferred with expanded blastocyst fresh IVP embryos. In summary, the effects of hCG or GnRH at ET on P/ET and calving/ET were inconsistent according to different embryo characteristics (e.g., embryo stage) and parity of recipients. Furthermore, treatment did not improve the overall fertility outcomes for recipient animals receiving IVP embryos.

Association between genomic daughter pregnancy rates and reproductive parameters in Holstein dairy cattle

The objective of this observational study was to evaluate the relationship between genomic daughter pregnancy rate (GDPR) with reproduction parameters such as pregnancy at first artificial insemination (AI), pregnancy per AI, and pregnancy losses (PL). A total of 12,949 events from 3,499 Holstein cows were included. Cows were enrolled as nulliparous (n = 1,220), primiparous (n = 1,314), or multiparous (n = 965). Cows were bred either after a timed AI protocol, timed embryo transfer (ET), or spontaneous estrus. Most lactating cows were bred following a timed AI protocol based on estradiol and progesterone, and most nulliparous were artificially inseminated following estrus detection. Hair samples were collected from the tail switch and cows were genotyped using a SNP platform (Clarifide, Zoetis). Cows that were bred by timed AI were evaluated for estrous behavior using tail chalk. Tail chalk was applied on the head of the tail 2 d before timed AI and the chalk was evaluated at AI (no estrus: 100% of chalk remaining or ≥50% of chalk remaining; Estrus: <50% of chalk remaining). Pregnancy diagnosis was performed at d 32 and 60 after AI using ultrasonography, and the presence of a heartbeat was considered a positive diagnosis. Pregnancy loss was defined as a pregnant cow on d 32 that was nonpregnant on d 60. As GDPR increased, the odds of pregnancy at first AI increased [odds ratio (OR) = 1.28, 95% CI = 1.20-1.35], the odds of pregnancy per AI increased (OR = 1.31, 95% CI = 1.25-1.36), and the odds of PL decreased (OR = 0.66, 95% CI = 0.60-0.72). Most cows that were bred on the day of the timed AI demonstrated estrus (n = 6,075; 92.9%). The odds of demonstrating estrus on the day of timed AI increased as GDPR increased (OR = 1.31, 95% CI = 1.17-1.48). There was no interaction between GDPR and parity or breeding management for pregnancy at first AI, pregnancy per AI, and PL. In conclusion, the odds of pregnancy at first AI and pregnancy per AI increased as GDPR increased. Moreover, the odds of PL increased as GDPR decreased. Greater GDPR was also associated with greater occurrence of estrus on the day of timed AI. These results suggest that selecting for higher GDPR could result in better reproductive performance, but this would need to be assessed with additional research.

Effect of gonadotropin-releasing hormone administered at the time of artificial insemination for cows detected in estrus by conventional estrus detection or an automated activity-monitoring system

The objectives of this study were to determine the effects of GnRH at the time of artificial insemination (AI) on ovulation, progesterone 7 d post-AI, and pregnancy in cows detected in estrus using traditional methods (tail chalk removal and mount acceptance visualization) or an automated activity-monitoring (AAM) system. We hypothesized that administration of GnRH at the time of AI would increase ovulation rate, plasma progesterone post-AI, and pregnancy per AI (P/AI) in cows detected in estrus. In experiment 1, Holstein cows (n = 398) were blocked by parity and randomly assigned to receive an injection of GnRH at the time of estrus detection/AI (GnRH, n = 197) or to remain untreated (control, n = 201) on 4 farms. The GnRH was administered as 100 µg of gonadorelin acetate. Ovarian structures and plasma progesterone were assessed in a subset of cows (GnRH, n = 52; control, n = 55) in experiment 1 at the time of AI and 7 d later. In experiment 2, a group of 409 cows in an AAM farm were enrolled as described for experiment 1 (GnRH, n = 207; control, n = 202). Data were categorized for parity (primiparous vs. multiparous), season (cool vs. warm), number of services (first vs. > first), DIM (>150 DIM vs. ≤150 DIM), and for AAM cows in experiment 2 for activity level (high: 90-100 index vs. low: 35-89 index). Pregnancy diagnosis was performed between 32 and 45 d post-AI (P1) and 60 to 115 d post-AI (P2). In experiment 1, there was no difference in plasma progesterone at day of estrus detection (control = 0.09 ng/mL vs. GnRH = 0.16 ng/mL), 7 d later (control = 2.03 ng/mL vs. GnRH = 2.18 ng/mL), and ovulation rate (GnRH = 83.2% vs. control = 77.9%) between treatments. There were no effects of GnRH in experiment 1 for P/AI at P1 (control = 43.3% vs. GnRH = 38.6%), P2 (control = 38.4% vs. GnRH = 34.5%), and for pregnancy loss (control = 9.8% vs. GnRH = 8.2%). In experiment 2, there were no effects of GnRH for P/AI at P1 (control = 39.6% vs. GnRH = 40.1%), P2 (control = 35.0% vs. GnRH = 37.4%), and for pregnancy loss (control = 9.5% vs. GnRH = 6.2%). There was a tendency for a parity effect on P/AI for P1, but not P2 or for pregnancy loss. High-activity cows had greater P/AI in P1 (low activity = 27.9% vs. high activity = 44.1%), P2 (low activity = 21.8% vs. high activity = 41.2%), and lower pregnancy loss (low activity = 20.7% vs. high activity = 5.1%), but there were no interactions between treatment and activity level. The current study did not support the use of GnRH at estrus detection to improve ovulatory response, progesterone 1 wk post-AI, and P/AI. More research is needed to investigate the relationship between GnRH at the time of AI and activity level in herds using AAM systems.

Occurrence and greater intensity of estrus in recipient lactating dairy cows improve pregnancy per embryo transfer

The aim of this study was to determine the association between occurrence and intensity of estrous expression with pregnancy success in recipient lactating dairy cows subjected to embryo transfer (ET). Two observational studies were conducted. Holstein cows were synchronized using the same timed ET protocol, based on estradiol and progesterone in both experiments. At 9 d after the end of the timed ET protocol only animals that had ovulated were implanted with a 7-d embryo [experiment 1 (Exp. 1); n = 1,401 ET events from 1,045 cows, and experiment 2 (Exp. 2); n = 1,147 ET events from 657 cows]. Embryos were produced in vivo (Exp. 1 and Exp. 2) and in vitro (only Exp. 2), then transferred to recipient cows as fresh or frozen-thawed. Pregnancy was confirmed at 29 and 58 d after the end of timed ET protocol. In Exp. 1, animals had their estrous expression monitored through a tail chalk applied on the tail head of the cows and evaluated daily for chalk removal (no estrus: 100% of chalk remaining; estrus: <50% of chalk remaining). In Exp. 2, cows were continuously monitored by a leg-mounted automated activity monitor. Estrous expression was quantified using the relative increase in physical activity at estrus in relation to the days before estrus. Estrous expression was classified as no estrus [<100% relative increase in activity (RI)], weak intensity (100-299% RI), and strong intensity (≥300% RI). Data were analyzed by analysis of variance using mixed linear regression models (GLIMMIX) in SAS (SAS Institute Inc.). A total of 65.2% (914/1,401) and 89.2% (1,019/1,142) of cows from Exp. 1 and Exp. 2, respectively, displayed estrus at the end of the ovulation synchronization protocol. In Exp. 1, cows expressing estrus before to ET had greater pregnancy per ET than those that did not [41.0 ± 2.3% (381/914) vs. 31.5 ± 2.9% (151/487), respectively]. Similarly, in Exp. 2, cows classified in the strong intensity group had greater pregnancy per ET compared with cows in the weak intensity and no estrus groups [41.3 ± 2.2% (213/571) vs. 32.7 ± 2.7% (115/353) vs. 11.3 ± 3.5% (26/218), respectively]. There was no effect of ET type on pregnancy per ET in Exp. 1. However, in Exp. 2, cows that received an in vivo-produced embryo, either fresh or frozen, had greater pregnancy per ET compared with cows that received in vitro-produced embryo. Cows receiving embryos in the early blastocyst and blastocyst stage had greater fertility compared with cows receiving embryos in the morula stage. There was an interaction between the occurrence of estrus and the stage of embryo development on pregnancy per ET, cows which displayed estrus and received a morula or early blastocyst had greater pregnancy per ET than cows that did not display estrus. In conclusion, the occurrence and the intensity of estrous expression improved pregnancy per ET in recipient lactating dairy cows and thus could be used as a tool to assist in the decision making of reproduction strategies in dairy farms.

164 Gonadotrophin-releasing hormone injection and colour flow Doppler ultrasound of the preovulatory follicle as a tool to increase pregnancy outcome after timed AI in beef cows

The aims of this study were to determine (1) the association between Doppler vascularisation scores (DVS) of the preovulatory follicle (POF) and fertility of beef cows submitted to timed AI (TAI) and (2) whether cows with low DVS benefit from a gonadotrophin-releasing hormone (GnRH) treatment at TAI. Multiparous lactating Nelore cows (Bos indicus; n=69) from a commercial beef farm in the state of Rondônia, Brazil, were enrolled in this study. Cows received 2mg of oestradiol benzoate intramuscularly (Bioestrogen, Biogénesis Bagó) and an intravaginal progesterone-releasing device (1.9g of progesterone; controlled internal drug release, CIDR) to synchronise follicular wave emergence on Day 0. The CIDR device was removed and cows were treated with 150μg of D-cloprostenol intramuscularly (prostaglandin F2α analogue; Croniben), 1mg of oestradiol cypionate intramuscularly, and 300IU of equine chorionic gonadotrophin (Novormon) intramuscularly on Day 8. Cows were then painted with a tail chalk marker to identify those displaying oestrus. All cows were submitted to TAI 48h after CIDR removal. At TAI, occurrence of oestrus was recorded and all cows were examined using transrectal ultrasonography. Blood flow of the POF was evaluated using colour Doppler imaging. Colour Doppler signals present on the follicular wall were subjectively scored using a 1-to-4 scale (1=absence or very low blood flow, and 4=intense blood flow detected on most of the follicular wall surface) adapted from Ginther (2007Ultrasonic Imaging and Animal Reproduction: Color-Doppler Ultrasonography, pages 87-114). Then, cows were divided into three groups according their DVS of the POF: (1) high DVS (DVS ≥3; n=36), (2) low DVS (DVS &amp;lt;3; n=16), and (3) low DVS (DVS &amp;lt;3; n=17) plus a GnRH treatment at TAI. The diameter of the POF was analysed using analysis of variance (PROC GLIMMIX of SAS; SAS Institute Inc.), and the means were compared among groups using Tukey's test. The proportion of cows that displayed oestrus and pregnancy rates was analysed using chi-square test. Cows in the high-DVS group had a larger POF than cows in the low-DVS and low-DVS-GnRH groups (13.2±0.2, 11.7±0.5, and 12.2±0.4, respectively; P&amp;lt;0.05). The proportion of cows that displayed oestrus was greater (P&amp;lt;0.05) in the high-DVS group (72%, 26/36) than in the low-DVS (37.5%, 6/16) or low-DVS-GnRH (53%, 9/17) groups. Finally, greater (P&amp;lt;0.05) pregnancy rates were observed in cows from the high-DVS (47.2%; 17/36) and low-DVS-GnRH (52.9%; 9/17) groups than in cows from the low-DVS group (18.7%; 3/16). The preliminary results from this study demonstrated that diameter of POF is positively associated with DVS. Moreover, cows that presented POF with higher DVS are more likely to become pregnant, and the administration of GnRH to females with low DVS can increase the fertility of beef cows submitted to TAI protocols. This study received funding support from Embrapa (MP1/PC3 project no. 01.03.14.011.00.00) and from CNPq (universal project no. 407307/2016-8).

155 Fertility of sexed semen in Holstein heifers and cows

Compared with conventional semen, use of sexed semen decreases the conception rate (CR); therefore, it is important to address the factors that lead to sperm damage. Recently, a novel sexed semen technology was developed for improving dairy cattle fertility (Betthauser et al. 2016 J. Dairy Sci. 99 (E-Suppl. 1), 534). However, there are few reports about the fertility of this novel sexed semen in dairy cattle in Japan. The objective of this study was to compare the CR of traditional sorting semen (S1: flow cytometry/cell sorting) and the novel sexed semen (S2: SexcelTM, ABS Global Inc.) in Holstein heifers and cows in Japan. The CR for the first insemination was obtained from 391 Holstein cows and 148 heifers (10.7-17.8 months old) from 14 dairy farms in Hokkaido from June 2017 to April 2019. Semen used for AI was collected from six bulls and packaged into 0.25-mL straws and frozen. Cows inseminated with conventional frozen semen (S3: unsexed semen in 0.5-mL straws) were used as controls. Calving data were collected from 123 cows and 69 heifers that had calved between January 2018 and May 2019. The diagnosis of pregnancy was confirmed using ultrasonography between 30 and 45 days after insemination. The CR and sex ratio were analysed using chi-square test. The average parity number was 1.9±1.1, and average days open was 84.9±20.4 days. The average interval between calving and the first service was 86.2±20.3 days. The average milk yield (at 305 days) was 12 195±1595 kg. All of the animals were inseminated after the onset of standing heat or removal of the tail chalk of natural heats and oestrus and ovulation synchronisation programmes on the farm. Conception rates of heifers of the first service of S1 (n=54) and S2 (n=94) were 68.5 and 70.2%, respectively. Conception rates of cows of the first service of S1 (n=32), S2 (n=173), and S3 (n=186) were 56.3, 57.8, and 53.8%, respectively. There were no significant differences in CR between these groups (P&amp;gt;0.4). The proportion of female calves produced using S1 (n=61) and S2 (n=57) was 91.8 and 93.0%, respectively, which was significantly more (P&amp;lt;0.01) compared with the 50.0% produced using S3 (n=74). There were no significant differences between S1 and S2 (P&amp;gt;0.8) in terms of the proportion of calves produced. The results of this study demonstrated that the traditional sorting and novel sexed semen (S1 and S2, respectively) did not differ in terms of the first-service CR and produced considerably more female calves than when the conventional semen (S3) was used. Furthermore, these findings indicate that the use of this novel sexed semen could achieve a sex ratio close to 90% female offspring.

Timed artificial insemination plus heat I: effect of estrus expression scores on pregnancy of cows subjected to progesterone–estradiol-based protocols

Expression of estrus near timed artificial insemination (TAI) is associated with greater fertility, and estrus detection could improve TAI fertility or direct TAI management, although accurate estrus detection can be difficult and time-consuming using traditional methods. The aim of this study is to evaluate influence of estrus on pregnancy (artificial insemination pregnancy rates (P/AI)) and to validate an alternative method to classify estrus/heat expression using tail chalking (HEATSC) in postpartum Bos indicus cows subjected to TAI in progesterone–estrogen-based protocols. In experiment 1 (Exp. 1), cows (5491) were subjected to visual observation of estrus after progesterone device removal, before TAI, and P/AI was evaluated according to estrus and body condition score (BCS). Cows received a progesterone device and 2 mg estradiol benzoate (EB). After 8 days, the device was removed and 150 μg of d-cloprostenol and 300 IU equine chorionic gonadotrophin was given. Later, animals in Exp. 1 received 1 mg EB and TAI 44 to 48 h. In the Exp. 2 – 3830 cows using similar protocol, received different ovulation inducers: 1 mg EB (n=1624) or 1 mg estradiol cypionate (EC; n=2206) on day 8 (D8). Cows were then marked with chalk, and HEATSC evaluated at TAI on D10 (HEATSC1 – no chalk removal=no estrus expression; HEATSC2 – partial chalk removal=low estrus expression; HEATSC3 – near complete/complete chalk removal=high estrus expression). In Exp. 1, cows showing estrus presented greater P/AI (48.4% v. 40.2%, P<0.05). In Exp. 2, P/AI (HEATSC1 – 40.0%; HEATSC2 – 49.7%; HEATSC3 – 60.9%; P<0.001), and larger follicle timed artificial insemination (LFTAI) (<0.001) varied according to HEATSC. There was no difference in P/AI (P=0.41) or LFTAI (P=0.33) according to ovulation inducer. Cows with greater BCS showed greater P/AI in both experiments (P<0.05). Estrus presence and greater HEATSC improved P/AI, and EC v. EB used promoted differential estrus manifestation (cows showing HEATSC2 and HEATSC3: 79.5% with EB v. 69.98% with EC use, P<0.001), however, with similar P/AI. The use of HEATSC in B. indicus cows subjected to TAI is useful to identify cows with greater estrus expression and consequently improved pregnancy rates in TAI, allowing the cows with low HEATSC to be targeted for additional treatments aimed at improving P/AI.

Timed artificial insemination plus heat II: gonadorelin injection in cows with low estrus expression scores increased pregnancy in progesterone/estradiol-based protocol

The use of tail chalk and estrus/heat expression scores (HEATSC) evaluation is instrumental in identifying cows with greater estrus expression and greater artificial insemination pregnancy rates (P/AI) in cows submitted to timed artificial insemination (TAI), and cows with low or no estrus expression present lower P/AI. It was intended in this study to improve the pregnancy rates in TAI for Bos indicus beef cows, and gonadotrophin-releasing hormone (GnRH) injection was hypothesized to increase pregnancy rates in a TAI program for cows submitted to progesterone–estradiol-based protocols with low or no estrus expression, evaluated by HEATSC. Cows (n= 2284) received a progesterone device and 2 mg estradiol benzoate, after 8 days the device was removed and 1 mg estradiol cypionate, 150 μg of d-cloprostenol and 300 IU equine chorionic gonadotropin was administered. All cows were marked with chalk and HEATSC evaluated (scales 1 to 3) at TAI performed on day 10. Animals with HEATSC1 and HEATSC2 (n= 937) received 100 μg de gonadorelin (GNRH group; n= 470), or 1 ml saline (Control group; n= 467), and cows with HEATSC3 (named HEAT group; n= 1347) received no additional treatment. The larger dominant follicle, evaluated on day 8and at TAI (day 10), was greater in HEAT group (P= 0.0145 and P <0.001, respectively). Corpus luteum (CL) area and progesterone concentration was evaluated on day 17, and CL area was larger in HEAT group, intermediary in Control and lower in GnRH group (Control= 2.68 cm2, GnRH= 2.37 cm2, HEAT group= 3.07 cm2, P <0.001). Greater progesterone concentrations were found in HEAT group than in Control and GnRH groups (Control= 4.74 ng/ml, GnRH= 4.29 ng/ml, HEAT group= 6.08 ng/ml, P<0.001). There was a difference in ovulation rate, greater in HEAT group than GnRH and Control groups (Control= 72.5%; GnRH= 81.25%; HEAT group= 90.71%; P= 0.0024). Artificial insemination pregnancy rates was greater in HEAT group (57.09% (769/1347) than in Control and GNRH groups, with positive effect of GnRH injection at the time of TAI in P/AI (Control= 36.18% (169/467), GnRH= 45.95% (216/470); P<0.0001). In conclusion, GnRH application in cows with low HEATSC (1 and 2) is a simple strategy, requiring no changes in TAI management to increase pregnancy rates in postpartum beef cows submitted to progesterone–estradiol-based TAI protocols, without reaching, however, the pregnancy rates of cows that demonstrate high estrus expression at the TAI.

11 Conception rate of high-producing dairy cows at first service using prostaglandin F2α and oestradiol benzoate

We have shown that a simplified oestrus synchronization protocol using prostaglandin F2α (PG) and oestradiol benzoate (EB) resulted in a satisfactory conception rate, similar to that obtained in high-producing dairy cows with natural oestrus (Yamaguchi et al. 2018 Reprod. Fertil. Dev. 30, 147 abstr). However, we found no significant effect of PG+EB protocols on the first-service conception rates (CR) of dairy cows. This study investigated the effect of the first-service CR by using PG+EB protocols in the high-producing dairy cows that had not exhibited oestrus expression after calving. Data concerning the first-service CR were obtained for 481 Holstein cows from 4 commercial dairy herds in Hokkaido and Ibaraki, Japan, from 2014 to 2017. The average number of parity, average interval between calving and the first service, and average milk yield (at 305 days) of the cows were 2.2±1.3, 89.7±26.1 days, and 10314±1994kg, respectively. We carried out the PG+EB protocols on cows not showing evident standing heat from Days 50 to 150 postpartum regardless of the presence of a corpus luteum in the ovary. The statuses of ovaries of cows were confirmed using ultrasonography before starting the treatment. Prostaglandin F2α (25 mg; Lutalyse, Pfizer, New York, NY, USA) was injected into cows with a &amp;gt;20mm cutoff for functional corpus luteum diameter at a random stage of oestrus, and EB (1 mg; Ovahormon®, Aska Animal Health Co., Tokyo, Japan) was injected 24h later, and AI was carried out 24 to 48h after EB injection. Cows with a natural oestrus service from Days 50 to 150 postpartum were used as control. All the cows were inseminated after the onset of standing heat or removal of the tail chalk. Pregnancy diagnoses were performed using ultrasonography 30 to 45 days after AI. The CR and intervals of partum to conception were analysed using a chi-square test and ANOVA. The first-service CR (i.e. the number of pregnant cows/total number of cows in the treatment group) of the natural oestrus (n=330) and PG+EB (n=151) groups were 42.7 and 41.1%, respectively, and there were no significant differences between these 2 groups (P=0.7). The average interval between calving and the first service of the PG+EB groups was significantly longer than that of the natural oestrus groups (86.4±1.5 days v. 96.9±1.9 days; P&amp;lt;0.01). The average days open of the natural oestrus and PG+EB groups were 88.5±3.2 days and 98.1±2.3 days, respectively (P&amp;lt;0.05). These results confirmed that the first-service CR in the PG+EB and natural oestrus groups of high-producing dairy cows were similar. We suggest that conducting PG+EB protocols was effective in the high-producing dairy cows that had not exhibited oestrus expression after calving. Moreover, using PG+EB protocols could prevent the further increase in the number of days open.

A description of interestrus and interservice intervals and associated fertility in 16 United Kingdom dairy herds

Understanding the changes that have occurred in reproductive management and physiology of the dairy cow is necessary to reverse the trend of declining fertility. In this study we analyzed the intervals between estrus events, as detected using tail chalk, in a population of 6,092 dairy cows from 16 herds in the central United Kingdom. Intervals were categorized by absence or presence of an insemination at the first estrus event, being designated interestrus intervals and interservice intervals, respectively. Modal interestrus interval was 21 d, as has been commonly described. Interservice interval was significantly longer than interestrus interval, with a modal value of 22 d. Multivariable analysis identified explanatory variables for intervals between estrus events, with random effects modeling accounting for clustering within herds and within individual cows. Intraclass correlation coefficients indicated that herd identity has a negligible effect, whereas animal identity accounts for 9% of interval variation. Parity, days in milk, insemination at the first estrus of the interval, and interval number within lactation all affected the interval. Logistic regression modeling indicated that interval length is also associated with the outcome of pregnancy. These findings indicate that intervals following an insemination are both longer than previously described and longer than interestrus intervals with no insemination event. The data also suggest that longer interservice interval length may be more conducive to subsequent conception than shorter intervals. Several factors appear to influence interestrus events and further work is required to investigate the underlying physiological mechanisms of these phenomena.

15 Effects of Timed Artificial Insemination Protocol Using Prostglandin F2α and Estradiol Benzoate on Conception Rate in High-Producing Dairy Cows

For several years, milk production per Holstein cow has been increasing while reproductive performance has declined worldwide. Timed AI (TAI) protocols with synchronization of oestrus and ovulation are tools for the improvement of reproductive performance in high producing dairy herds. In protocols for synchronization of oestrus and ovulation, oestradiol benzoate (EB) has been used to increase the number of animals that show oestrous behaviour, synchronize ovulation, and improve conception rate (CR). The objective of this study was to compare a simple oestrus synchronization and TAI protocol using prostaglandin F2α (PG) and EB with a more complicated TAI protocol or oestrus detection and AI on CR in high-producing Holstein cows. The study was conducted from 2014 to 2016, using 1,036 Holstein cows from 3 commercial dairy herds in central Hokkaido, Japan. The average parity number was 2.6 ± 1.5, and days open averaged 152.2 ± 82.0 days. The average milk yield (305 days) was 11,348 ± 1,619 kg. Oestrus synchronization of cows was achieved using PG followed by EB and TAI (PG was given to cows with a cutoff for CL diameter of &gt;20 mm by ultrasonography; EB (1 mg) was administered 24 h later and AI was carried out 24 to 48 h after EB injection), or by Heatsynch + CIDR [gonadotropin-releasing hormone (GnRH) injection and CIDR insertion at random stages of the oestrous cycle, CIDR removal, and PG injection 7 days later, EB injected 24 h after CIDR removal, and TAI 24 to 32 h after EB injection]. Cows observed in natural oestrus were used as controls. All cows were inseminated after the onset of standing heat or removal of the tail chalk (Paintstik®, LA-CO Industries, Elk Grove Village, IL, USA). Pregnancy diagnoses were performed using ultrasonography between 30 and 45 days after AI. The CR were compared using chi-square test. The CR (no. of pregnant cows/no. of total cows in the treatment group) of the natural oestrus group, PG + EB, and Heatsynch + CIDR cows were 39.2% (302/771), 38.7% (48/124), and 31.2% (44/141), respectively. Overall CR for the 3 groups was 38.0%. There were no significant differences in CR among the 2 groups (P &gt; 0.07). These results show that a simple oestrus synchronization protocol with injection of PG followed by EB can be used effectively for high-producing dairy herds, yielding a satisfactory CR similar to that obtained with the Heatsynch + CIDR protocol or natural oestrus.

The effect of tail paint formulation and heifer behavior on estrus detection

Although heifers can have better conception rates than cows, they are still subject to poor estrus detection and economic losses from reduced reproductive efficiency. Tail paint has been successful in identifying estrus, but behaviors such a licking or rubbing have been believed to remove the paint and lead to false-positives. To investigate tail paint utilization and potential relationships among behaviors, eighteen Holstein heifers were randomly assigned to one of three treatments: a control tail chalk (CON), tail chalk with proprietary ingredient (CHALK+); and a spray formulation (SPRAY). Experimental design was a replicated 3×3 Latin square. Visual observations were performed in 30min segments every 2h from 6AM to 6PM. Ovaries were examined via ultrasound imaging on d 0, 7, and 9 of each period. The presence of follicles or a corpus luteum (CL) was recorded with their respective sizes. Heifers receiving SPRAY had a lower number of licks received per day and less tail paint removed regardless of day or follicle size when compared with CON or CHALK+. Rump lick received, chin rest received, anogenital sniff received, mount received, and both initiated and received behaviors for attempt to mount occurred more in heifers with large follicles regardless of day. Producers looking for heifers to breed should focus on those receiving rump lick, chin resting, anogenital sniff, mount, and attempt to mount. The use and combination of these estrus detection tools can improve reproductive efficiency in dairy operations.

109 COMPARISON OF METHODS FOR SYNCHRONIZING RECIPIENTS OF IN VITRO PRODUCED EMBRYOS

The present study compared fertility, as pregnancy per embryo transfer (P/ET), and efficiency of recipient utilisation, as pregnancy per treated potential recipient (P/TX), in heifers receiving in vitro-produced embryos using synchronized oestrus after prostaglandin F2α (OESTRUS) or synchronized ovulation and fixed timed embryo transfer (FTET) with new or reused CIDR. In Expt. 1, heifers (n = 520) were randomly assigned to 1 of 3 groups: OESTRUS, FTET with new CIDR, or FTET with second-use CIDR (previously used for 5 days). Heifers in OESTRUS group (n = 166) were synchronized with two prostaglandin F2α 14 days apart and detection of oestrus performed using tail chalk during 5 days after the second prostaglandin F2α. Heifers in FTET were synchronized with a new CIDR (n = 178) or second-use CIDR (n = 176) using a modified 5-day CIDR-Synch; Day –8: CIDR inserted; Day –3: CIDR removed, prostaglandin F2α; Day –2: second prostaglandin F2α; Day 0: gonadotropin-releasing hormone to induce ovulation. In Expt. 2, heifers (n = 422) were randomly assigned to 1 of 2 groups: FTET with new CIDR or FTET with third-use CIDR (previously used twice for 5 days each time) using the FTET protocol described for Expt. 1. Fresh in vitro-produced embryos were transferred between 6 and 8 days after OESTRUS or gonadotropin-releasing hormone (FTET). All heifers were evaluated by transrectal ultrasonography on Day 32 and 60 for pregnancy detection. Measurements of P/ET and P/TX for both experiments were analysed by logistic regression (LOGISTIC procedure, SAS 9.4) using biologically meaningful covariates such as embryo stage and quality, interval from oestrus or GnRH to transfer, and technician in the statistical analyses. In Expt. 1, two preplanned contrasts were performed to compare differences between OESTRUS v. FTET, and between FTET with new v. second-use CIDR. The P/ET at Day 32 was similar (P = 0.50) with 41.3% (45/109) for OESTRUS and 43.4% (134/309) for FTET groups. Similarly, P/ET on Day 60 was 30.3% (33/109) for OESTRUS and 32.4% (100/309) for FTET groups (P = 0.37). However, P/TX heifer on Day 60 was greater (P = 0.04) in the FTET (28.2%; 100/354) compared to OESTRUS (19.9%; 33/166). This difference is attributed to a greater (P &lt; 0.001) utilisation efficiency (transferred/treated) of heifers in FTET (87.3%) v. OESTRUS (65.6%). In the second contrast, P/ET on Day 32 were similar (P = 0.87) for FTET heifers synchronized with a new CIDR (43.9%, 69/157) v. second-use CIDR (42.8%, 65/152). In addition, P/TX heifer on Day 60 was also similar (P = 0.52) for heifers receiving a new or second-use CIDR (29.8%, 53/178 v. 26.7% 47/176). In Expt. 2, P/ET on Day 32 was similar (P = 0.73) for FTET with a new CIDR (41.0%, 77/188) or third-use CIDR (42.3%, 83/196). The P/TX heifer on Day 60 was also not different (P = 0.58) for new CIDR (28.6%, 61/213) v. third-use CIDR (31.1%, 65/209). Thus, use of FTET with new or used CIDR can produce similar P/ET and greater efficiency of recipient utilisation compared to OESTRUS.

Effect of intrauterine dextrose on reproductive performance of lactating dairy cows diagnosed with purulent vaginal discharge under certified organic management

The objectives of the study were to assess responses to treatments (clinical cure and resumption of estrous cycles) of cows with purulent vaginal discharge (PVD) that received intrauterine infusion of a hypertonic solution of 50% dextrose (DEX) or untreated control (CON) cows and the subsequent pregnancy per artificial insemination (PAI) in cows with and without PVD. Cows (n=2,852) from 2 dairy herds were screened for PVD using the gloved hand technique at exam 1 [26±3d in milk (DIM)]. Cows with vaginal discharge scores of 2 or 3 (0–3 scale) were stratified by parity and randomly allocated into 1 of 2 treatment groups: (1) intrauterine infusion (~200mL) of 50% DEX solution (n=456), or (2) untreated control animals (CON, n=491). Fourteen days posttherapy (40±3 DIM), cows with PVD were re-examined at exam 2 (40±3 DIM) to assess the response to treatments. All cows were subjected to the same reproductive program, which consisted of estrus detection twice daily (using tail chalking and visual observation) for the first 5 artificial inseminations; then, open lactating cows were turned out with bulls. Cows displaying signs of standing estrus underwent AI and no reproductive hormones were used. Pregnancy diagnosis (PD) was performed via transrectal palpation at 40±3d post-AI. The risk of culling within 14d posttherapy was not different among treatment groups. Cows with PVD had greater cervical diameter at exam 1 and decreased PAI compared with cows without PVD. Treatment with DEX increased the proportion of cows with clear vaginal discharge (clinical cure) and cyclicity 14d posttherapy compared with CON cows. Pregnancy per AI for DEX (29.2±2%) cows was significantly greater than that for CON (22.5±2%) cows. Cows without PVD had a greater proportion of cycling cows (65.6%) and PAI (37%) with reduced pregnancy losses (5.7%) compared with DEX or CON cows. The use of intrauterine DEX alone improved reproductive performance of cows with PVD.

Progesterone supplementation to lactating dairy cows without a corpus luteum at initiation of the Ovsynch protocol

The objectives were to determine the effect of progesterone supplementation on fertility responses in lactating dairy cows without corpora lutea (CL) at initiation of the timed artificial insemination (AI) program. Holstein cows from 5 commercial dairy farms were subjected to the Ovsynch-56 protocol (d −10 GnRH, d −3 PGF2α, d −0.7 GnRH, d 0 AI). Ovaries were scanned by ultrasonography on d −10. Within farm, cows without CL were blocked by pen and assigned randomly to remain as nonsupplemented controls (CON; n=652) or to receive 2 controlled internal drug-release (CIDR) inserts containing 1.38g of progesterone each from d −10 to −3 (2CIDR; n=642). Cows with CL were randomly selected within pen and used as positive controls as cows in diestrus at the initiation of the Ovsynch protocol (DIEST; n=640). Signs of estrus were detected beginning on d −9 based on removal of tail chalk, and cows in estrus received AI on the same day. Blood samples from subsets of cows on d −10, −9, −7, −5, −3, and 0 (n=109) and on d 6, 13, and 19 (n=156) were analyzed for progesterone concentrations. Pregnancy was diagnosed on d 32 and 60 after AI. The average progesterone concentration during the timed AI program was lowest for CON, intermediate for 2CIDR, and highest for DIEST (0.92, 2.77, and 4.93 ng/mL, respectively). The proportions of cows that ovulated in response to the first GnRH (63.6, 61.1, and 47.2%, respectively) and that had a new CL on d −3 at PGF2α injection (72.4, 67.9, and 47.4%, respectively) were greater for CON and 2CIDR compared with DIEST, respectively. The diameter of the ovulatory follicle and the proportion of cows that ovulated in response to the second GnRH did not differ among treatments. A greater proportion of CON and 2CIDR cows were detected in estrus at AI compared with DIEST cows (35.8, 39.6, and 30.6%, respectively). Pregnancy per AI was less for CON compared with 2CIDR and DIEST on d 32 (31.3, 42.2, and 38.4%, respectively) and d 60 after AI (28.9, 37.2, and 33.9%, respectively), indicating that progesterone supplementation reestablished fertility in cows lacking a CL similar to that of cows in diestrus at the initiation of the timed AI program. Treatment did not affect pregnancy loss between d 32 and 60 of gestation. Pregnancy from a subset of cows with plasma progesterone concentrations indicated that a minimum concentration of 2.0 ng/mL was needed to optimize fertility. A single ultrasound examination effectively identified a low-fertility cohort of cows based on the absence of CL at the first GnRH injection of the Ovsynch protocol. Supplementation with 2 CIDR inserts increased progesterone in plasma by an additional 1.85 ng/mL compared with CON, resulting in concentrations of 2.77 ng/mL during development of the ovulatory follicle, which restored fertility in dairy cows lacking CL to a level similar to that of cows in diestrus.

Effect of progesterone supplementation on fertility responses of lactating dairy cows with corpus luteum at the initiation of the Ovsynch protocol

The objectives of this study were to determine the effects of supplemental progesterone on fertility responses of lactating dairy cows with a CL at the initiation of the timed artificial insemination (AI) program. Holstein cows were subjected to the Ovsynch protocol (Day −10 GnRH, Day −3 PGF2α, Day −0.7 GnRH, and Day 0 timed AI). Ovaries were scanned by ultrasonography on Day −10 and cows with CL were blocked by pen and assigned randomly to receive no supplemental progesterone (control, n = 863) or to receive a controlled internal drug-release (CIDR) insert containing progesterone from Days −10 to −3 (1CIDR, n = 862). Cows were observed for signs of estrus beginning on Day −9 based on removal of tail chalk and those in estrus received AI on the same day. Blood sampled from a subset of cows was analyzed for progesterone concentrations on Days −10, −9, −7, −5, −3, 0, 6, 13, and 19. Pregnancy was diagnosed on Days 32 and 60 after AI. Supplementation increased (P < 0.01) progesterone concentrations between Day −9 and −3 compared with control (7.5 vs. 6.2 ng/mL). Treatment had no effect on the ovulatory response to the first and final GnRH injections of the Ovsynch protocol, the proportion of cows that maintained their CL until the day of PGF2α injection, or the diameter of the ovulatory follicle before AI. Although the overall proportion of cows in estrus at AI did not differ between treatments, progesterone supplementation prevented (P < 0.01) cows from coming into estrus from Days −9 to −3 (0.0% vs. 4.7%), whereas it increased (P = 0.02) the proportion of cows inseminated in estrus from Days −2 to −1 (7.1% vs. 4.5%). Pregnancy per AI (P/AI) tended to be reduced (P = 0.06) by supplemental progesterone on Day 32 (40.5% vs. 45.0%), but not on Day 60 after AI (36.6% vs. 39.7%). A tendency for an interaction (P = 0.09) between treatment and the presence of CL at the PGF2α injection was observed for P/AI on Day 32 in cows that received timed AI because progesterone supplementation reduced P/AI in cows that maintained their CL until Day −3 (40.3% vs. 46.7%); however, it increased P/AI in those that did not have a CL at PGF2α (38.1% vs. 27.7%). Treatment did not affect pregnancy loss between Days 32 and 60 of gestation. In conclusion, incorporating a single intravaginal insert to the timed AI program increased progesterone concentrations in plasma by 1.3 ng/mL, but did not benefit fertility in dairy cows that have CL at the initiation of the synchronization protocol.

Evaluation of a contract breeding management program in Ohio dairy herds: Test day summary and economic measures

A field study was conducted to assess the impact of a contract breeding program that was offered by a breeding co-operative and featured tail chalking and daily evaluation of cows for insemination by co-operative technicians; dairy employees no longer handled estrous detection and insemination activities. From early 2002 until mid-2004, herd-level test day summary records related to production and reproduction were obtained for 32 herds identified as well-managed client herds of the breeding co-operative. Using analyses that controlled for other predictors and random herd-level effects, average days to first service were less by 13 days ( P = 0.0037) and estrous detection rate was greater by 12% ( P = 0.0011) for program than for non-program herds. Although first service conception rate was slightly less and the program herds used 0.34 more services per conception ( P = 0.1488) than non-program herds, the program herds averaged 16 fewer days before pregnancy ( P = 0.028). Test day summary information and representative estimates of feed, milk, and semen prices were used in a spreadsheet-based model to estimate a partial budget annuity value for an average cow in each herd on each test day. Value of an average cow from a contract herd did not significantly differ from a non-contract herd, even though the analyses suggested an economic benefit for the program herds; the modeling did not, however, account for costs of the program implementation. Additional analyses did not find any significant associations between technician and on days to first service, first service conception rate, estrous detection rate, services per conception, or days open.

Evaluation of a contract breeding management program in selected Ohio dairy herds with event-time analysis: II. Parametric frailty models

The effect of a contract breeding program offered by a breeding co-operative was assessed using parametric frailty models with event-time analysis technique in a field study of Ohio dairies. The program featured tail chalking and daily evaluation of cows for insemination by co-operative technicians; dairy employees no longer handled estrus detection activities. Test day records were obtained between early 2002 and mid-2004 for 16,453 lactations representing 11,398 cows in 31 herds identified as well-managed client herds by the breeding co-operative. Various parametric distributions for event times available in a commercial software (Stata 9.1, College Station, TX) were tested to assess which distribution fit the calving-to-conception data best. After identifying the distribution with the best fit, a full model with potential confounders and other significant predictors of time to pregnancy was developed and then frailty terms were included in the model. Generalized gamma and log-normal distributions fit the data best, but since gamma distribution does not allow the use of frailty effects, log-normal distribution was used in further modeling. Separate accelerated failure time models with frailty terms to account for latent effects at the herd, cow, or lactation level were developed, testing both gamma and inverse Gaussian frailty distributions. In these models, potential confounders and statistically significant predictors were also controlled for, and the association between the contract breeding program and the mean time to pregnancy was characterized using time ratios. The log-normal model identified that interval to pregnancy was associated with breed, herd size, use of ovulation synchronization protocols, parity, calving season and somatic cell score (above or below 4.5) and maximum milk yield prior to pregnancy or censoring. While controlling for these factors, there was a reduction in average time to pregnancy among cows managed under the contract breeding program. All frailty terms were highly significant, regardless of whether it was an individual frailty at the lactation level or a shared frailty at the cow or herd level, suggesting that there was considerable heterogeneity within these levels. Inclusion of a frailty term at the herd level changed the estimate for the contract breeding program considerably, while a frailty term on other levels did not, indicating that herd characteristics (e.g., overall management) have a substantial impact on reproductive performance and should be accounted for in the analysis. Interpretation using time ratios with or without a shared herd frailty found that the contract breeding program was associated with a reduction of 6.5% and 14.1% in mean time to pregnancy, respectively.

Evaluation of a contract breeding management program in selected Ohio dairy herds with event-time analysis: I. Cox proportional hazards models

An observational study was conducted in order to assess the impact of a contract breeding program on the reproductive performance in a selected group of Ohio dairies using event-time analysis. The contract breeding program was offered by a breeding co-operative and featured tail chalking and daily evaluation of cows for insemination by co-operative technicians. Dairy employees no longer handled estrus detection activities. Between early 2002 and mid-2004, test-day records related to production and reproduction were obtained for 16,453 lactations representing 11,398 cows in a non-random sample of 31 dairies identified as well-managed client herds of the breeding co-operative. Of the 31 herds, 15 were using the contract breeding at the start of the data acquisition period, having started in the previous 2 years. The remaining 16 herds managed their own breeding program and used the co-operative for semen purchase. Cox proportional hazards modeling techniques were used to estimate the association of the contract breeding, as well as the effect of other significant predictors, with the hazard of pregnancy. Two separate Cox models were developed and compared: one that only considered fixed covariates and a second that included both fixed and time-varying covariates. Estimates of effects were expressed as the hazard ratio (HR) for pregnancy. Results of the fixed covariates model indicated that, controlling for breed, herd size, use of ovulation synchronization protocols in the herd, whether somatic cell score exceeded 4.5 prior to pregnancy or censoring, parity, calving season, and maximum test-day milk prior to pregnancy or censoring, the contract breeding program was associated with an increased hazard of pregnancy (HR = 1.315; 95% CI 1.261–1.371). The results of the time-varying covariates model, which controlled for breed, herd size, use of ovulation synchronization protocols, somatic cell score above 4.5, parity, calving season, and testing season also found that the program was associated with an increased hazard of pregnancy (HR = 1.387; 95% CI 1.327–1.451). The fixed and time-varying covariates models both found similar sets of predictors when analyzing the association of the contract breeding program with hazard of pregnancy. Both models identified a 30% or greater increase in hazard of pregnancy associated with use of the contract breeding program, suggesting that herds subscribing to the program achieved pregnancies in a more timely fashion.